Il-12 immunotherapy for cancer

ABSTRACT

Compositions and methods for delivering immune modulatory molecules to result in a therapeutic effect are disclosed. The compositions and methods use stably integrating lentiviral delivery systems. The methods are useful for therapeutically and prophylactically treating cancer such as leukemia.

RELATED APPLICATIONS

The present application is a continuation of copending U.S. patentapplication Ser. No. 12/598,899, filed Nov. 4, 2009, which is a Nationalstage entry of International Application No. PCT/CA2008/000849, filedMay 5, 2008, which claims priority to U.S. Provisional PatentApplication 60/916,136 filed May 4, 2007, each of these applicationsbeing incorporated herein in their entirety by reference.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the Sequence Listing“10723-P4537US04_SL.txt” (72,365 bytes), submitted via EFS-WEB andcreated on May 21, 2014, is herein incorporated by reference.

FIELD OF INVENTION

The invention relates generally to compositions and methods fortherapeutically and prophylactically treating cancer. In particular, thepresent invention pertains to IL-12, lentiviral vectors encoding IL-12for transducing cells and use of the transduced cells for cancerimmunotherapy.

BACKGROUND OF THE INVENTION

Cancer immunotherapy aims to overcome the inability of the immune systemto efficiently protect against the establishment of tumors or rejectestablished tumors.

Lentiviral Vectors (LVs)

Lentiviral vectors (LVs) are efficient gene transfer agents. They arestable and can be concentrated by ultracentrifugation to high titers.Compared to adenovirus, for example, they generate little immuneconsequences on their own reducing responses against transduced cells.Advances in LV design, safety, and long-term testing will increase theirclinical adaptation. LVs have been used in cancer immunogene therapy(Metharom, P. et al., 2001; Firat, H. et al., 2002), the induction ofDCs (Esslinger, C. et al., 2003) and antigen presentation for CTLresponses (Breckpot, K. et al., 2003; Esslinger, C. et al., 2003), andthe transduction of CD34+ cells differentiated into DCs towards HIV/AIDSimmunotherapy DCs (Gruber, A. et al., 2003).

Interleukin-12

Cancer cells express antigens. Despite the presence of such antigens,tumors are generally not readily recognized and eliminated by the host,as evidenced by the development of disease. The inability of the immunesystem to protect against tumors may be due to mechanisms of evasion,active suppression, or sub-optimal activation of the response.

Cytokines are integral to both the innate and acquired immune systems.They can alter the balance of cellular and humoral responses, alterclass switching of B lymphocytes and modify innate responses.

Interleukin-12 is a heterodimeric cytokine with multiple biologicaleffects on the immune system. It is composed of two subunits, p35 andp40, both of which are required for the secretion of the active form ofIL-12, p70. Interleukin-12 acts on dendritic cells (DC), leading toincreased maturation and antigen presentation, which can allow for theinitiation of a T cell response to tumor specific antigens. It alsodrives the secretion of IL-12 by DCs, creating a positive feedbackmechanism to amplify the response. Once a response is initiated, IL-12plays a fundamental role in directing the immune system towards a Th1cytokine profile, inducing CD4⁺ T cells to secrete interferon-gamma(IFN-γ) and leading to a CD8⁺ cytotoxic T cell response.⁴ However, IL-12is also a strong pro-inflammatory cytokine that leads to the secretionof other cytokines including tumor necrosis factor-alpha (TNF-α) which,combined with IFN-γ, is a prerequisite for the development of CD4′cytotoxic T lymphocytes (CTL).⁵ Furthermore, IL-12 can promote theactivation of innate immune cells such as macrophages and eosinophilsthrough its induction of IFN-γ and other cytokines. This activation thenleads to IL-12 secretion by these cells and further amplification ofboth the innate and acquired responses.⁴ However, high levels of IL-12,and consequently IFN-γ, have also been associated with induction ofantagonistic molecules such as IL-10 and the depletion of signallingmolecules downstream of IL-12, such as STAT4.⁶⁻⁸

Direct injection of recombinant IL-12 has been shown in some mousemodels of leukemia.⁹⁻¹³ While initial human trials employing thisapproach were less promising (14-17 discussed in 4).

Innovative gene therapy strategies may accelerate the development ofprophylactic immunotherapy against cancer.

SUMMARY

The inventors have demonstrated that intraperitoneal (IP) administrationof low dose rIL-12 elicits a protective response against an establishedtumor burden and that this CD⁸⁺ T cell-dependent response leads tolong-term immune memory. The inventors also delivered IL-12 by way oftransduced tumor cells, mediated by a lentiviral delivery system toensure that optimum concentrations of IL-12 were available at the tumorsite. The method of delivering IL-12 is highly effective and is readilyapplied to a variety of cancers.

The application provides in one aspect, a composition comprising:

a lentiviral vector;an IL-12 expression cassette.

In one embodiment, the IL-12 expression cassette comprises apolynucleotide optionally encoding a p35 polypeptide and apolynucleotide encoding a p40 polypeptide; or a polynucleotide encodingan IL-12 fusion polypeptide. In another embodiment the IL-12 fusionpolypeptide has at least 70% sequence identity to SEQ ID NO: 4 and bindsan IL-12 receptor. In a further embodiment, the lentiviral vectoroptionally comprises one or more of a: 5′-Long terminal repeat (LTR),HIV signal sequence, HIV Psi signal 5′-splice site (SD), delta-GAGelement, Rev Responsive Element (RRE), 3′-splice site (SA), Elongationfactor (EF) 1-alpha promoter and 3′-Self inactivating LTR (SIN-LTR). Inyet a further embodiment, the lentiviral vector comprises a centralpolypurine tract optionally SEQ ID NO:2 and/or a woodchuck hepatitisvirus post-transcriptional regulatory element, optionally SEQ ID NO:3;or a sequence having at least 70% sequence identity to SEQ ID NO:2and/or SEQ ID NO:3. In another embodiment, the lentiviral vectorcomprises a pHR′ backbone. In one embodiment, the lentiviral vector is aclinical grade vector. In one embodiment, the composition furthercomprises an activator polynucleotide encoding a polypeptide thatconverts a prodrug to a drug, optionally a modified tmpk polynucleotide.In yet a further embodiment, the activator polynucleotide comprises atmpk polynucleotide with at least 80% sequence identity to a modifiedtmpk polynucleotide described herein.

In certain embodiments, the composition further comprises a detectioncassette. In one embodiment, the detection cassette comprises a CD19,truncated CD19, CD20, human CD24, murine HSA, human CD25 (huCD25), atruncated form of low affinity nerve growth factor receptor (LNGFR),truncated CD34, eGFP, eYFP, or any other fluorescent protein orerythropoietin receptor (EpoR) polynucleotide; or a polynucleotide withat least 70% sequence identity to said polynucleotide.

In another embodiment, the composition further comprises an immunemodulatory cassette. In one embodiment, the immune modulatory cassettecomprises a polynucleotide that encodes a polypeptide that modulates animmune cell, optionally a dendritic cell or a T cell, optionally a CD4+T cell, optionally CD40L, IL-7, or IL-15. In another embodiment thecomposition is a pharmaceutical composition and further comprises apharmaceutically acceptable carrier.

In another aspect, the application provides a vector constructcomprising:

a lentiviral vector;an IL-12 expression cassette.

In another aspect the application provides an isolated virus comprisingthe vector construct or composition described herein.

A further aspect provides an isolated cell secreting IL-12 at the orabove the threshold level, wherein the cell is optionally transducedwith the composition, the vector construct or the isolated virusdescribed herein. In one embodiment, the cell is a cancer cell,optionally an established cell line, optionally a primary cancer cell,optionally a cancer cell derived from a subject. In another embodiment,the cancer cell is a leukemic cell, optionally an ALL cell, an AML cell,a CML cell or a CLL cell. In a further embodiment, the threshold levelis at least 1500 pg/mL/10⁶ cells/2 hrs of IL-12, optionally at least1500 pg/mL/10⁶ cells/2 hrs, 1500-2500 pg/mL/10⁶ cells/2 hrs, 2500-5000pg/mL/10⁶ cells/2 hrs, 5000-7500 pg/mL/10⁶ cells/2 hrs, 7500-10000pg/mL/10⁶ cells/2 hrs, 10000-12500 pg/mL/10⁶ cells/2 hrs, 12500-15000pg/mL/10⁶ cells/2 hrs, 15000-17500 pg/mL/10⁶ cells/2 hrs, 17500-20000pg/mL/10⁶ cells/2 hrs or at least 20000 pg/mL/10⁶ cells/2 hrs of IL-12

Another aspect provides a population of cells comprising isolated cellsand/or transduced cells described herein wherein the population of cellsoptionally comprises at least 0.1 to 1% IL-12 producing cells,optionally leukemic cells, optionally about 0.5%, about 1%, about 1-5%,5-10%, 10-20% or more IL-12 producing cells, optionally leukemic cells,and wherein the population of cells secretes above the threshold leveloptionally the threshold level necessary to induce or enhance a CD4+ Tcell dependent immune response, optionally at least 1500 pg/mL/10⁶cells/2 hrs, 1500-2500 pg/mL/10⁶ cells/2 hrs, 2500-5000 pg/mL/10⁶cells/2 hrs, 5000-7500 pg/mL/10⁶ cells/2 hrs, 7500-10000 pg/mL/10⁶cells/2 hrs, 10000-12500 pg/mL/10⁶ cells/2 hrs, 12500-15000 pg/mL/10⁶cells/2 hrs, 15000-17500 pg/mL/10⁶ cells/2 hrs, 17500-20000 pg/mL/10⁶cells/2 hrs or at least 20000 pg/mL/10⁶ cells/2 hrs of IL-12. In oneembodiment, the population of cells is derived from a clone thatsecretes IL-12 above the threshold level optionally at least 1500pg/mL/10⁶ cells/2 hrs of IL-12.

A further aspect provides a composition comprising the isolated virus,cell or population of cells described herein.

Another aspect of the disclosure provides a method of expressing IL-12in a cell, optionally a cancer cell comprising contacting the cell withthe composition, the vector construct or the isolated virus underconditions that permit transduction of the cell, thereby providing atransduced cell, optionally wherein the IL-12 is secreted. In oneembodiment, the method further comprises a step of isolating thetransduced cell or isolating a population of cells comprising thetransduced cell. In another embodiment, the method further comprises:

growth arresting the transduced cell, the population of cells orcomposition; andintroducing the transduced cell, population of cells and/or compositionin a subject.

Another aspect provides a method of reducing the number of tumor cellsor cancer burden in a subject in need thereof comprising administeringto the subject an isolated virus, transduced cell, population of cellsor composition described herein. Another aspect provides a method oftreating a subject with cancer or an increased risk of cancer comprisingadministering to the subject an isolated virus, transduced cell,population of cells or composition described herein. In certainembodiments, the method further comprises monitoring cancer progression.

In certain embodiments, the cancer is a solid tumor. In otherembodiments, the cancer is leukemia, optionally ALL, AML, CML or CLL.

A further aspect provides a method of inducing or enhancing an immuneresponse in a subject optionally with cancer or an increased risk ofcancer comprising administering t administering to the subject anisolated virus, transduced cell, population of cells or compositiondescribed herein.

In one aspect the application provides a method of inducing or enhancinga memory immune response in a subject, optionally with cancer or anincreased risk of cancer, comprising administering to the subject anisolated virus, transduced cell, population of cells or compositiondescribed herein. In certain embodiments, the immune response comprisesa CD4+ T cell mediated immune response. In certain embodiments, thetransduced cell is growth arrested prior to administering to thesubject. In one embodiment, the transduced cell is irradiated prior toadministering to the subject.

Also provided, is a method of delivering IL-12 to a subject, optionallywith cancer or an increased risk or cancer, optionally, for enhancingcancer treatment comprising:

generating an IL-12 secreting cell wherein IL-12 secreted per cell isabove a threshold level; andintroducing an effective number of the generated IL-12 secreting cellsto the subject.

Another aspect provides a method of sustaining IFNgamma levels inducedby IL-12 in a host comprising:

generating an IL-12 secreting cell wherein IL-12 secreted per cell isabove a threshold level; andintroducing an effective number of the generated IL-12 secreting cellsto the patient.

In certain embodiments, the threshold level of IL-12 secreted is atleast 1.5 fg/ml/cell/2 hrs. In other embodiments, the threshold level ofIL-12 secreted is at least 1.5 pg/ml cells/2 hrs. In certainembodiments, the IL-12 secreting cell is generated by contacting thecell with a composition comprising a lentiviral delivery vector and anIL-12 expression cassette.

In certain embodiments, the cell is optionally a cancer cell, optionallyderived from the subject with cancer. In certain embodiments, the cellsare introduced by IP injection, subcutaneously or intradermally.

In certain embodiments, the immune response is initiated against aleukemia. In certain embodiments, the immune response is initiatedsubstantially free of inducing or enhancing of a CD⁸⁺ T cell-dependentimmune response. In certain other embodiments, the immune response leadsto long-term immune memory. In certain embodiments, the immune responsedoes not induce or enhance antagonistic cytokines.

In certain embodiments, the level of IL-12 produced is above a thresholdlevel that enhances dendritic cell maturation and/or antigenpresentation.

In another aspect, the application provides use of an isolated virus,transduced cell, population of cells or composition described herein forreducing the number of tumor cells or cancer burden in a subject in needthereof.

In another aspect the application provides use of an isolated virus,transduced cell, population of cells or composition described herein fortreating a subject with cancer.

In another aspect the application provides use an isolated virus,transduced cell, population of cells or composition described herein forinducing or enhancing an immune response in a subject.

In another embodiment, the application provides use an isolated virus,transduced cell, population of cells or composition described herein forinducing or enhancing a memory immune response in a subject.

In another aspect the application provides use of an IL-12 secretingcell for delivering IL-12 to a subject, optionally with cancer or anincreased risk of cancer optionally for enhancing cancer treatment:

generating an IL-12 secreting cell wherein IL-12 secreted per cell isabove a threshold level; andisolating an effective number of the generated IL-12 secreting cells forintroduction to the subject.

In another aspect the application provides use of an isolated virus,transduced cell, population of cells or composition described herein,for treating a subject in need thereof, optionally a subject with canceror an increased risk of developing cancer.

In certain embodiments, the number of cells administered ranges from 10⁵cells to 10⁹ cells, optionally about 10⁵, about 10⁶ cells, about 10⁷cells, about 10⁸ cells, or about 10⁹ cells. In other embodiments, thepopulation of cells administered ranges from 10⁵ cells to 10⁹ cells,optionally about 10⁵ cells, about 10⁶ cells, about 10⁷, cells, about 10⁸cells, or about 10⁹ cells.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

The following non-limiting examples are illustrative of the presentinvention:

FIG. 1 a IP administered rIL-12-mediated protection of mice challengedwith 70Z13-L cells. Mice were challenged with 10⁶ cells IP and receivedeither no treatment-or injections of 0.1, 1, 10 or 20 ng/mouse/dayrIL-12 for 14 days (n=5 mice for each group).

FIG. 1 b IP administered rIL-12 therapy leads to long-term protectionagainst challenge with 70Z13-L. (i) Naïve mice (A, n=10) were challengedwith 70Z/3-L cells on day 0 and treated for 14 days with injections of20 ng rIL-12/mouse/day. A group of mice (B₁, n=7) were included ascontrols for the 70Z/3-L cells (curve comparison by log rank testp=0.001). (ii) After a period of 70 days, five mice from group A, havingundergone rIL-12 therapy, were secondarily challenged with 10⁶ 70Z/3-Lcells without further rIL-12 treatment. The other five animals were keptto confirm that no toxicity appeared after 70 days. Five naïve mice (B₂)were included to demonstrate the lethality of the 70Z/3-L cells(comparison of Kaplan-Meier survival curves was performed using Logranktest p=0.0015).

FIG. 1 c Delayed IP administration of rIL-12 therapy leads toprotection. Mice were injected with 10⁴ 70Z/3-L cells on day 0. Acontrol group (70Z/3-L) did not receive treatment (n=4). From days 0through 5, groups of 4 or 5 mice (5 mice for days 0, 1 and 2, 4 mice fordays 3, 4 and 5) started receiving injections of 20 ng rIL-12/mouse/dayfor 14 days. Animals were monitored and euthanized at the appearance ofsymptoms. Curve comparison was performed using Logrank test. Alltreatment groups are significantly different from the control group(p=0.0029) but are not significantly different from each other.

FIG. 1 d Requirement of T cells and IFN-γ for rIL-12-mediated protectionfollowing IP administration. Mice (n=5 mice in each group) were depletedusing antibodies as described in Materials and Methods. The mice werechallenged with 10⁶ 70Z/3-L cells IP, injected with 20 ng/mouse/dayrIL-12 and monitored for the appearance of symptoms. Comparison ofKaplan-Meier survival curves was performed using Logrank test(p<0.0018).

FIG. 2 a Schematic representation of the LV-muIL-12(LV-cPPT-EF1-mIL-12-WPRE) vector. LTR: long-terminal repeat; SD: splicedonor; RRE; rev response element; SA: splice acceptor; cPPT: centralpolypurine tract; CMV: cytomegalovirus; WPRE: woodchuck hepatitis virusposttranscriptional regulatory element; muIL-12: murine interleukin-12;SIN: self-inactivating LTR.

FIG. 2 b Interleukin-12 secretion by vector-transduced clones is astable trait. Levels of IL-12 secretion were measured by ELISA on 2-5independent occasions and seen to remain fairly constant; differencesare not statistically significant.

FIG. 3 Leukemia cell mediated IL-12 therapy leads to protection ofchallenged mice. Mice were injected IP with PBS or 10⁶ cells of eitherthe parent line, 70Z/3-L, or one of the vector-transduced clones andmonitored for the appearance of symptoms. Clones secrete varying levelsof IL-12 and a theoretical threshold was established, below whichprotection is not conferred.

FIG. 4 Leukemia cell mediated IL-12 therapy leads to protection ofchallenged mice when only a portion of the cells are vector-transduced.Mice were injected IP with 10⁶ cells of the parent line, 70Z/3-L, andvarying proportions a.) 2%, 10% and 50% of the LV12.1 secreting clone orb.) 0.1%, 0.5%, 1% and 10% of LV12.2 and LV12.3, and monitored for theappearance of symptoms.

FIG. 5 Leukemia cell mediated IL-12 therapy leads to long-term andspecific protection against challenge with 70Z/3-L. Mice were initiallychallenged with either 10⁶ LV12.2 cells or injected with PBS. More than110 days following the primary challenge, primed mice (n=4 in eachgroup) were secondarily challenged with either 10⁶ 70Z/3-L or 10⁶ L1210cells. The PBS injected mice (n=5 in each group) also received either10⁶ 70Z/3-L or 10⁶ L1210 cells to control for their efficiency to leadto morbidity, or another injection of PBS and monitored for appearanceof symptoms. Kaplan-Meier survival curve comparison was performed usingLogrank test, p<0.0001.

FIG. 6 Requirement of the CD4⁺ T cell subset for leukemia cell-mediatedprotection of challenged mice. Mice (n=5 in each group) were depletedusing antibodies as described in Materials and Methods. The mice werechallenged with 10⁶ LV12.2 cells IP and monitored for the appearance ofsymptoms. Kaplan-Meier curve comparison was performed using Logranktest, p=0.0084.

FIG. 7 Cytokine expression profiles of mice receiving IP administrationand leukemia cell-mediated IL-12 therapies. The mice (n=4 in each group)receiving IP administered rIL-12 therapy were challenged with 10⁶70Z/3-L cells and received either no treatment or injections of 10 or 20ng/mouse/day rIL-12 for 14 days. Mice (n=4 in each group) receivingleukemia cell-mediated IL-12 therapy were challenged with 10⁶ 70Z/3-Lcells IP and received either no treatment or treatment with variousproportions (0.5%, 1% or 10%) of the vector-transduced clone LV12.2.Serum samples were collected and analyzed on days 7, 10 and 20 asdescribed in Materials and Methods. (*—all mice from group 2 in theleukemia cell-mediated model were dead by day 20 such that serum was notcollected from this group).

FIG. 8 a mIL-12 expression in transduced and transfected AML lines.

FIG. 8 b hIL-12 expression in transduced and transfected 293T cells.

FIG. 8 c hIL-12 expression in AML.1 cells.

FIG. 9 Anti-CD122 antibody increases human cell engraftment in NOD/SCIDmice. NOD/SCID mice were either not pre-treated (n=3) or pre-treatedwith anti-CD122 (200 μg; i.p. injection; n=3). 24 hrs later, mice wereirradiated (350 cGy; ¹³³Cs source) and injected i.v. with 7×10⁵ purifiedcord blood-derived human CD34⁺ cells. At 7 weeks post-transplant, bonemarrow was harvested, and human cell engraftment was determined by flowcytometry using anti-human CD45 PE. Two of three control recipientslacked long-term human cell engraftment, as defined by ≦1% CD45⁺ events.

DETAILED DESCRIPTION

The inventors have shows that administration of low dose recombinantIL-12 (rIL-12) elicits a protective response against an establishedleukemia burden and that rejection is mediated by a CD4⁺ and CD8⁺ Tcell-dependent immune response which leads to long-term immune memorywithout the induction of antagonistic cytokines. The inventors havecompared this protocol to a cell therapy approach in which leukemiccells were transduced with a lentivirus vector (LV) engineeringexpression of murine IL-12 (both subunits) cDNA. Clones of the leukemiccells producing a wide range of IL-12 were established. Injection ofIL-12 producing leukemic cells provoked long term and specific immunitywithout the induction of antagonistic mechanisms. Leukemia clearance inthis instance, however, was mediated by a CD4⁺ cellular subset alone,suggesting a qualitatively different route to immunity than that seen insystemic therapy. The inventors found that injection of as few as 1%IL-12 producing leukemic cells along with 99% untransduced leukemiccells, was sufficient to elicit protective immunity as long as each ofthese cells produced IL-12 above a necessary threshold. This finding mayexplain the failure of many human cell therapy based protocols becausein these cases IL-12 production is measured on bulk populations makingit impossible to know if sufficient IL-12 is being produced in the localenvironment influenced by the IL-12 producing cell. The averageproduction reported in these studies is well below the thresholdreported in the present disclosure.

The vector constructs, compositions, cells and methods described hereinfor delivering IL-12 are highly effective and are readily applied to avariety of cancers.

DEFINITIONS

The term “a cell” as used herein includes a plurality of cells.

The term “ALL” as used herein refers to acute lymphoblastic leukemia isa rapidly growing leukemia wherein the malignant hematopoietic cells arelymphoid precursor cells. Cytogenetic abnormalities occur in ˜70% ofcases of ALL in adults but are not associated with a singletranslocation event.

The term “allogenic” also referred to as “allogeneic” as used hereinmeans cells, tissue, DNA, or factors taken or derived from a differentsubject of the same species. For example in the context where allogenictransduced cancer cells are administered to a subject with cancer,cancer cells removed from a patient that is not the subject, aretransduced or transfected with a vector that directs the expression ofIL-12 and the transduced cells are administered to the subject. Thephrase “directs expression” refers to the polynucleotide comprising asequence that encodes the molecule to be expressed. The polynucleotidemay comprise additional sequence that enhances expression of themolecule in question.

The term “AML” as used herein refers to acute myeloid leukemia, arapidly progressing disease in which too many immature non-lymphocytewhite blood cells are present in the blood and bone marrow. Also calledacute myelogenous leukemia, acute myeloblastic leukemia, acutenonlymphocytic leukemia, and ANLL.

The term “antibody” as used herein is intended to include monoclonalantibodies, polyclonal antibodies, and chimeric antibodies. The antibodymay be from recombinant sources and/or produced in transgenic animals.The term “antibody fragment” as used herein is intended to includewithout limitations Fab, Fab′, F(ab′)2, scFv, dsFv, ds-scFv, dimers,minibodies, diabodies, and multimers thereof, multispecific antibodyfragments and Domain Antibodies. Antibodies can be fragmented usingconventional techniques. For example, F(ab′)2 fragments can be generatedby treating the antibody with pepsin. The resulting F(ab′)2 fragment canbe treated to reduce disulfide bridges to produce Fab′ fragments. Papaindigestion can lead to the formation of Fab fragments. Fab, Fab′ andF(ab′)2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecificantibody fragments and other fragments can also be synthesized byrecombinant techniques. The term also includes antibodies or antibodyfragments that bind to the detecting cassette polypeptides disclosedherein.

By “at least moderately stringent hybridization conditions” it is meantthat conditions are selected which promote selective hybridizationbetween two complementary nucleic acid molecules in solution.Hybridization may occur to all or a portion of a nucleic acid sequencemolecule. The hybridizing portion is typically at least 15 (e.g. 20, 25,30, 40 or 50) nucleotides in length. Those skilled in the art willrecognize that the stability of a nucleic acid duplex, or hybrids, isdetermined by the Tm, which in sodium containing buffers is a functionof the sodium ion concentration and temperature (Tm=81.5° C.−16.6 (Log10 [Na+])+0.41 (%(G+C)−600/l), or similar equation). Accordingly, theparameters in the wash conditions that determine hybrid stability aresodium ion concentration and temperature. In order to identify moleculesthat are similar, but not identical, to a known nucleic acid molecule a1% mismatch may be assumed to result in about a 1° C. decrease in Tm,for example if nucleic acid molecules are sought that have a >95%identity, the final wash temperature will be reduced by about 5° C.Based on these considerations those skilled in the art will be able toreadily select appropriate hybridization conditions. In preferredembodiments, stringent hybridization conditions are selected. By way ofexample the following conditions may be employed to achieve stringenthybridization: hybridization at 5× sodium chloride/sodium citrate(SSC)/5×Denhardt's solution/1.0% SDS at Tm−5° C. based on the aboveequation, followed by a wash of 0.2×SSC/0.1% SDS at 60° C. Moderatelystringent hybridization conditions include a washing step in 3×SSC at42° C. It is understood, however, that equivalent stringencies may beachieved using alternative buffers, salts and temperatures. Additionalguidance regarding hybridization conditions may be found in: CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in:Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold SpringHarbor Laboratory Press, 2001.

The term “autologous” as used herein refers to cells, tissue, DNA orfactors taken or derived from an individual's own tissues, cells or DNA.For example in the context where autologous transduced cancer cells areadministered to a subject with cancer, cancer cells removed from thesubject are transduced or transfected with a vector that directs theexpression of IL-12 and the transduced cells are administered to thesubject.

The phrase “cancer burden” refers to the quantum of cancer cells orcancer volume in a subject. Reducing cancer burden accordingly refers toreducing the number of cancer cells or the cancer volume in a subject.

The phrase “cancer that is characterized by periods of remission” referto cancers that may respond to a treatment but wherein the cancer recursat some later time suggesting that not all cancer cells were eradicatedby the treatment. An example of such a cancer is CLL.

The term “cancer cell” as used herein refers to any cell that is acancer cell or is derived from a cancer cell e.g. clone of a cancercell.

The term “cassette” as used herein refers to a polynucleotide sequencethat is to be expressed. The cassette can be inserted into a vector. Thecassette optionally includes regulatory sequence to direct or modify itsexpression.

The phrase “cell surface protein” or “cell surface polypeptide” as usedherein refers to a polypeptide that is expressed, in whole or in part onthe surface of a cell. This optionally includes polypeptide fragmentsthat are presented on cells as well as polypeptides or fragments thereofthat are naturally found on the surface of a cell. In the context of acell modified to express a vector construct comprising a detectioncassette polypeptide, wherein the detection cassette polypeptide is acell surface polypeptide, the cell surface marker need not be native tothe cell it is being expressed on.

The term “CLL” refers to chronic lymphocytic leukemia, a slow growingtype of leukemia. CLL is the most common leukemia of adults with anexpectation of ˜16500 cases in North America in 2008. Remissions can beachieved with purine analogues and monoclonal antibody therapy howeverthe diseases invariable progresses. CLL is also referred to as chroniclymphoblastic leukemia. B-CLL is a subset of CLL.

The term “clinical grade vector” as used herein refers to a vectormanufactured using near-GMP or GMP procedures and quality assurancetested.

The term “CML” refers to chronic myeloid leukemia, a slowly progressingleukemia wherein excessive white blood cells are made in the bonemarrow. The hallmark of this disease is the reciprocal translocationbetween chromosomes 9 and 22 leading to the formation of the Bcr-Abloncogene. This is manifested by a rapid expansion of bone marrow-derivedhematopoietic cells of the myeloid lineage. CML is also referred to aschronic myelogenous leukemia, and chronic granulocytic leukemia.

A “conservative amino acid substitution” as used herein, is one in whichone amino acid residue is replaced with another amino acid residuewithout abolishing the protein's desired properties. Conservative aminoacid substitutions are known in the art. For example, conservativesubstitutions include substituting an amino acid in one of the followinggroups for another amino acid in the same group: alanine (A), serine(S), and threonine (T); aspartic acid (D) and glutamic acid (E);asparagine (N) and glutamine (Q); arginine (R) and lysine (L);isoleucine (I), leucine (L), methionine (M), valine (V); andphenylalanine (F), tyrosine (Y), and tryptophan (W).

The term “detection cassette” as used herein refers to a polynucleotidethat directs expression of a molecule that is useful for enriching,sorting, tracking and/or killing cells in which it is expressed. Thedetection cassette encodes a polypeptide that is expressed in thetransduced or transfected cell and can as a result be used to detectand/or isolate transduced or transfected cells. The detection cassetteis optionally used to determine the efficiency of cell transduction ortransfection.

As used herein, the phrase “effective amount” or “therapeuticallyeffective amount” or a “sufficient amount” of composition, vectorconstruct, virus or cell of the present application is a quantitysufficient to, when administered to the subject, including a mammal, forexample a human, effect beneficial or desired results, includingclinical results, and, as such, an “effective amount” or synonym theretodepends upon the context in which it is being applied. For example, inthe context of treating cancer, it is an amount of the composition,vector construct, virus or cell sufficient to achieve a treatmentresponse as compared to the response obtained without administration ofthe composition, vector construct, virus or cell The amount of a givencompound of the present application that will correspond to such anamount will vary depending upon various factors, such as the givenagent, the pharmaceutical formulation, the route of administration, thetype of disease or disorder, the identity of the subject (e.g. age, sex,weight) or host being treated, and the like, but can nevertheless beroutinely determined by one skilled in the art. Also, as used herein, a“therapeutically effective amount” of a composition, vector construct,virus or cell of the present disclosure is an amount which results in abeneficial or desired result in a subject as compared to a control. Asdefined herein, a therapeutically effective amount of a composition,vector construct, virus or cell of the present disclosure may be readilydetermined by one of ordinary skill by routine methods known in the art.Dosage regime may be adjusted to provide the optimum therapeuticresponse.

The term “hybridize” refers to the sequence specific non-covalentbinding interaction with a complementary nucleic acid.

An “immune modulatory cassette” as used herein, means a polynucleotidethat directs expression of a molecule or polypeptide that enhances theanti-tumor effect of an IL-12 transduced cell. One class of immuneregulatory molecules is cytokines. Also included are compounds thatinhibit molecules that antagonize IL-12 response. For example, IL-10 caninhibit IL-12, compounds that inhibit the antagonistic effect of IL-10would positively modulate the immune response.

The term “immune response” as used herein can refer to activation ofeither or both the adaptive and innate immune system cells such thatthey shift from a dormant resting state to a state in which they areable to elaborate molecules typical of an active immune response.

The phrase “inducing an immune response” as used herein refers to amethod whereby an immune response is activated. The phrase “enhancing animmune response” refers to augmenting an existing but immune response.

The term “increased risk of cancer” as used herein means a subject thathas a higher risk of developing a particular cancer than the averagerisk of the population. A subject may have a higher risk due topreviously having had said particular cancer and or having a geneticrisk factor for said particular cancer.

The term “kills” with respect to transfected or transduced cells refersto inducing cell death through any of a variety of mechanisms includingapoptosis, necrosis and autophagy. For example an agent that iscytotoxic kills the cells.

The term “leukemia” as used herein refers to any cancer or precanceroussyndrome that initiates in blood forming tissues such as the bonemarrow. A number of leukemias have been characterized including ALL,AML, CLL, and CML. Delivery of a LV/IL-12 construct to engineer IL-12expression in dendritic cells or other efficient antigen-presentingcells could also be effective in a pre-cancerous state if dominanttumor-associated antigens had been identified for the future cancer inthat case and the host immune response re-directed against that antigen.

The term “polynucleotide” and/or “nucleic acid sequence” as used hereinrefers to a sequence of nucleoside or nucleotide monomers consisting ofnaturally occurring bases, sugars and intersugar (backbone) linkages.The term also includes modified or substituted sequences comprisingnon-naturally occurring monomers or portions thereof. The nucleic acidsequences of the present application may be deoxyribonucleic acidsequences (DNA) or ribonucleic acid sequences (RNA) and may includenaturally occurring bases including adenine, guanine, cytosine,thymidine and uracil. The sequences may also contain modified bases.Examples of such modified bases include aza and deaza adenine, guanine,cytosine, thymidine and uracil; and xanthine and hypoxanthine.

The term “polypeptide” as used herein refers to a sequence of aminoacids consisting of naturally occurring residues, and non-naturallyoccurring residues.

The term “promoter” as used herein refers to a recognition site on DNAthat is bound by an RNA polymerase. The polymerase drives transcriptionof the transgene.

The term “sequence identity” as used herein refers to the percentage ofsequence identity between two polypeptide sequences or two nucleic acidsequences. To determine the percent identity of two amino acid sequencesor of two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of afirst amino acid or nucleic acid sequence for optimal alignment with asecond amino acid or nucleic acid sequence). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=number of identical overlappingpositions/total number of positions.times.100%). In one embodiment, thetwo sequences are the same length. The determination of percent identitybetween two sequences can also be accomplished using a mathematicalalgorithm. A preferred, non-limiting example of a mathematical algorithmutilized for the comparison of two sequences is the algorithm of Karlinand Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modifiedas in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A.90:5873-5877. Such an algorithm is incorporated into the NBLAST andXBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLASTnucleotide searches can be performed with the NBLAST nucleotide programparameters set, e.g., for score=100, wordlength=12 to obtain nucleotidesequences homologous to a nucleic acid molecules of the presentapplication. BLAST protein searches can be performed with the XBLASTprogram parameters set, e.g., to score-50, wordlength=3 to obtain aminoacid sequences homologous to a protein molecule of the presentinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., 1997, NucleicAcids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to performan iterated search which detects distant relationships between molecules(Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, thedefault parameters of the respective programs (e.g., of XBLAST andNBLAST) can be used (see, e.g., the NCBI website). The percent identitybetween two sequences can be determined using techniques similar tothose described above, with or without allowing gaps. In calculatingpercent identity, typically only exact matches are counted.

The term “subject” as used herein includes all members of the animalkingdom including mammals, suitably humans including patients.

The term “subject in need thereof” refers to a subject that couldbenefit from the method, and optionally refers to a subject with cancer,such as leukemia, or optionally a subject with increased risk of cancer,such as a subject previously having cancer, a subject with aprecancerous syndrome or a subject with a strong genetic disposition.

The term “transduction” as used herein refers to a method of introducinga vector construct or a part thereof into a cell. Wherein the vectorconstruct is comprised in a virus such as for example a lentivirus,transduction refers to viral infection of the cell and subsequenttransfer and integration of the vector construct or part thereof intothe cell genome.

The term “treating” or “treatment” as used herein means administering toa subject a therapeutically effective amount of the compositions, cellsor vector constructs of the present application and may consist of asingle administration, or alternatively comprise a series ofapplications.

As used herein, and as well understood in the art, “treatment” or“treating” is also an approach for obtaining beneficial or desiredresults, including clinical results. Beneficial or desired clinicalresults can include, but are not limited to, alleviation or ameliorationof one or more symptoms or conditions, diminishment of extent ofdisease, stabilized (i.e. not worsening) state of disease, preventingspread of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment. Further any of the treatment methods or uses described hereincan be formulated alone or for contemporaneous administration with otheragents or therapies.

The term “vector construct” as used herein means a recombinantpolynucleotide comprising a vector alternatively referred to as a vectorbackbone and at least one coding cassette. A vector construct isoptionally comprised in a virus, such as a lentivirus. The term “vector”has used herein refers to a means by which polynucleotides can beintroduced into a cell or host.

Vector Constructs and Virus

The application provides in one aspect a vector construct or virus suchas a lentivirus comprising a delivery vector and and IL-12 expressioncassette. In one embodiment the delivery vector is a lentivirus orlentiviral vector (LV) backbone.

Interleukin-12 (IL-12) Expression Cassette

Interleukin-12 is a heterodimeric cytokine with multiple biologicaleffects on the immune system. It is composed of two subunits, p35 andp40, both of which are required for the secretion of the active form ofIL-12, p70. Interleukin-12 acts on dendritic cells (DC), leading toincreased maturation and antigen presentation, which can allow for theinitiation of a T cell response to tumor specific antigens.

In one embodiment the IL-12 expression cassette comprises apolynucleotide that directs expression of IL-12 polypeptide. Any IL-12polypeptide including variants and derivatives of known IL-12 moleculescan be used. In a preferred embodiment, the IL-12 is human IL-12. Inanother embodiment, the IL-12 is murine IL-12.

In one embodiment the polynucleotide comprises the sequence of bothIL-12 subunits, p35 and p40, separated by an IRES sequence which permitsexpression of multiple transgenes from a single transcript. In otherembodiments, the polynucleotide directs expression of an IL-12 fusionpolypeptide that retains IL-12 activity. In one embodiment, thepolynucleotide that directs the expression of IL-12 comprises a cDNAencoding a human IL-12 polypeptide fusion obtained from InVivoGen (pORFwith IL-12elasti(p40::p35)). In one embodiment, the polynucleotidedirects the expression of an IL-12 polypeptide comprising all or part ofSEQ ID NO:4 or 5, and/or a variant of a fragment thereof that retainsIL-12 activity. In another embodiment, the polynucleotide directsexpression of an IL-12 fusion polypeptide that has at least 70%, 70-80%,80-90%, 90-95%, 95-99.9% or more to the IL-12 portion of SEQ ID NO:4 or5 and retains IL-12 activity. IL-12 activity is determined for exampleby assessing activation of the IL-12 receptor in a cell based assay.

A person skilled in the art will understand that non-critical residuescan be deleted, and or mutated without effect on IL-12. Polynucleotidesdirecting expression of IL-12 polypeptide analogs are also contemplated.

Delivery Vectors

It will be appreciated by one skilled in the art that a variety ofdelivery vectors and expression vehicles are usefully employed tointroduce a modified DNA molecule into a cell. Vectors that are usefulcomprise lentiviruses, oncoretroviruses, expression plasmids,adenovirus, and adeno-associated virus. Other delivery vectors that areuseful comprise herpes simplex viruses, transposons, vaccinia viruses,human papilloma virus, Simian immunodeficiency viruses, HTLV, humanfoamy virus and variants thereof. Further vectors that are usefulcomprise spumaviruses, mammalian type B retroviruses, mammalian type Cretroviruses, avian type C retroviruses, mammalian type D retroviruses,HTLV/BLV type retroviruses, and lentiviruses.

Vectors such as those listed above have been employed to introduce DNAmolecules into cells for use in gene therapy. Examples of vectors usedto express DNA in cells include vectors described in: Kanazawa T,Mizukami H, Okada T, Hanazono Y, Kume A, Nishino H, Takeuchi K, KitamuraK, Ichimura K, Ozawa K. Suicide gene therapy using AAV-HSVtk/ganciclovirin combination with irradiation results in regression of human head andneck cancer xenografts in nude mice. Gene Ther. 2003 January;10(1):51-8. Fukui T, Hayashi Y, Kagami H, Yamamoto N, Fukuhara H, TohnaiI, Ueda M, Mizuno M, Yoshida J Suicide gene therapy for human oralsquamous cell carcinoma cell lines with adeno-associated virus vector.Oral Oncol. 2001 April; 37(3):211-5.

Retroviral Vectors

In one embodiment, the delivery vector is a retroviral vector. In afurther embodiment, the delivery vector is a lentiviral vector.Lentiviral vectors (LVs), a subset of retroviruses, transduce a widerange of dividing and non-dividing cell types with high efficiency,conferring stable, long-term expression of the transgene²⁵⁻²⁷.

The use of lentivirus-based gene transfer techniques relies on the invitro production of recombinant lentiviral particles carrying a highlydeleted viral genome in which the transgene of interest is accommodated.In particular, the recombinant lentivirus are recovered through the intrans coexpression in a permissive cell line of (1) the packagingconstructs, i.e., a vector expressing the Gag-Pol precursors togetherwith Rev (alternatively expressed in trans); (2) a vector expressing anenvelope receptor, generally of an heterologous nature; and (3) thetransfer vector, consisting in the viral cDNA deprived of all openreading frames, but maintaining the sequences required for replication,incapsidation, and expression, in which the sequences to be expressedare inserted.

In one embodiment the lentiviral vector comprises one or more of a5′-Long terminal repeat (LTR), HIV signal sequence, HIV Psi signal5′-splice site (SD), delta-GAG element, Rev Responsive Element (RRE),3′-splice site (SA), Elongation factor (EF) 1-alpha promoter and 3′-Selfinactivating LTR (SIN-LTR). The lentiviral vector optionally comprises acentral polypurine tract (cPPT; SEQ ID NO: 2) and a woodchuck hepatitisvirus post-transcriptional regulatory element (WPRE; SEQ ID NO: 3). In afurther embodiment, the lentiviral vector comprises a pHR′ backbone. Incertain embodiments, the pHR′ back bone comprises for example asprovided below.

In one embodiment the Lentigen lentiviral vector described in Lu, X. etal. Journal of gene medicine (2004) 6:963-973 is used to express the DNAmolecules and/or transduce cells.

In one embodiment the lentiviral vector comprises a 5′-Long terminalrepeat (LTR), HIV signal sequence, HIV Psi signal 5′-splice site (SD),delta-GAG element, Rev Responsive Element (RRE), 3′-splice site (SA),Elongation factor (EF) 1-alpha promoter and 3′-Self inactivating LTR(SIN-LTR). It will be readily apparent to one skilled in the art thatoptionally one or more of these regions is substituted with anotherregion performing a similar function.

In certain embodiments the IL-12 is required to be expressed atsufficiently high levels. Transgene expression is driven by a promotersequence. Optionally, the lentiviral vector comprise a CMV promoter. Inanother embodiment, the promoter is Elongation factor (EF) 1-alphapromoter. A person skilled in the art will be familiar with a number ofpromoters that will be suitable in the vector constructs describedherein.

Enhancer elements can be used to increase expression of modified DNAmolecules or increase the lentiviral integration efficiency. In oneembodiment the lentiviral vector further comprises a nef sequence. In apreferred embodiment the lentiviral further comprises a cPPT sequencewhich enhances vector integration. The cPPT acts as a second origin ofthe (+)-strand DNA synthesis and introduces a partial strand overlap inthe middle of its native HIV genome. The introduction of the cPPTsequence in the transfer vector backbone strongly increased the nucleartransport and the total amount of genome integrated into the DNA oftarget cells. In an alternate preferred embodiment, the lentiviralvector further comprises a Woodchuck Posttranscriptional RegulatoryElement (WPRE). The WPRE acts at the transcriptional level, by promotingnuclear export of transcripts and/or by increasing the efficiency ofpolyadenylation of the nascent transcript, thus increasing the totalamount of mRNA in the cells. The addition of the WPRE to lentiviralvector results in a substantial improvement in the level of transgeneexpression from several different promoters, both in vitro and in vivo.In a further preferred embodiment, the lentiviral vector comprises botha cPPT sequence and WPRE sequence. In yet a further embodiment, thelentiviral vector comprises a sequence having at least 70%, 70-80%,80-90%, 90-95%, 95-99.9% or more sequence identity to SEQ ID NO:2 and/orSEQ ID NO:3. The vector also comprises in an alternate embodiment aninternal ribosome entry site (IRES) sequence that permits the expressionof multiple polypeptides from a single promoter.

In addition to IRES sequences, other elements which permit expression ofmultiple polypeptides are useful. In one embodiment the vector comprisesmultiple promoters that permit expression more than one polypeptide. Inanother embodiment the vector comprises a protein cleavage site thatallows expression of more than one polypeptide. Examples of proteincleavage sites that allow expression of more than one polypeptidecomprise those listed in the following articles which are incorporatedby reference: Retroviral vector-mediated expression of HoxB4 inhematopoietic cells using a novel coexpression strategy. Klump H,Schiedlmeier B, Vogt B, Ryan M, Ostertag W, Baum C. Gene Ther. 200;8(10):811-7; A picornaviral 2A-like sequence-based tricistronic vectorallowing for high-level therapeutic gene expression coupled to adual-reporter system Mark J. Osborn, Angela Panoskaltsis-Mortari, Ron T.McElmurry, Scott K. Bell, Dario A. A. Vignali, Martin D. Ryan, Andrew C.Wilber, R. Scott Mclvor, Jakub Tolar and Bruce R. Blazar. MolecularTherapy 2005; 12 (3), 569-574; Development of 2A peptide-basedstrategies in the design of multicistronic vectors. Szymczak A L,Vignali DA. Expert Opin Biol Ther. 2005; 5(5):627-38; Correction ofmulti-gene deficiency in vivo using a single ‘self-cleaving’ 2Apeptide-based retroviral vector. Szymczak A L, Workman C J, Wang Y,Vignali K M, Dilioglou S, Vanin E F, Vignali D A. Nat Biotechnol. 2004;22(5):589-94. It will be readily apparent to one skilled in the art thatother elements that permit expression of multiple polypeptides whichidentified in the future are useful and may be utilized in the vectorsof the invention.

In certain embodiments, the lentiviral vector is a clinical gradevector.

Viral Regulatory Elements

The viral regulatory elements are components of delivery vehicles usedto introduce nucleic acid molecules into a host cell. The viralregulatory elements are optionally retroviral regulatory elements. Forexample, the viral regulatory elements may be the LTR and gag sequencesfrom HSC1 or MSCV. The retroviral regulatory elements may be fromlentiviruses or they may be heterologous sequences identified from othergenomic regions.

One skilled in the art would also appreciate that as other viralregulatory elements are identified, these may be used with the nucleicacid molecules of the invention.

Detection Cassette

In certain embodiments, the vector construct comprises a detectioncassette. The detection cassette comprises a polynucleotide that directsexpression of a molecule that is useful for enriching, sorting, trackingand/or killing cells in which it is expressed. The detection cassetteencodes a polypeptide that is expressed in the transduced or transfectedcell and can as a result be used to detect and/or isolate transduced ortransfected cells. The detection cassette is optionally used todetermine the efficiency of cell transduction or transfection.

In one embodiment, the detection cassette encodes a polypeptide thatprotects from a selection drug such as neomycin phosphotransferase orG418. In another embodiment, the detection cassette encodes afluorescent protein such as GFP. Other fluorescent proteins can also beused. In a further embodiment, the detection cassette is a cell surfacemarker such as CD19, truncated CD19, CD20, human CD24, murine HSA, humanCD25 (huCD25), a truncated form of low affinity nerve growth factorreceptor (LNGFR), truncated CD34 or erythropoietin receptor (EpoR). Incertain embodiments the detection cassette polypeptide is substantiallyoverexpressed in transduced cells such that these cells arepreferentially targeted. In other embodiments, the detection cassettepolypeptide is not appreciably expressed on the cell type to betransduced or transfected.

As described below, the detection cassette polypeptide can be used toisolate transduced cells by methods such as flow cytometry.

In one embodiment, the detection cassette comprises a CD19 molecule orfragment thereof. In another preferred embodiment the constructcomprises a detection polynucleotide incorporated intopHR′-cppt-EF-IRES-W-SIN, pHR′-cppt-EF-huCEA-IRES-hCD19-W-SIN orpHR′-cppt-EF-HER/neuIRES-hCD19-W-SIN. Additionally it will be readilyapparent to one skilled in the art that optionally one or more of theseelements can be added or substituted with other regions performingsimilar functions.

Immune Modulatory Cassette

Enhanced antitumor effect is obtainable with the use of specific immunemodulatory molecules. One class of immune regulatory molecules iscytokines. Cytokines are integral to both the innate and acquired immunesystems. They can alter the balance of cellular and humoral responses,alter class switching of B lymphocytes and modify innate responses.

In one embodiment, the immune modulatory cassette comprises apolynucleotide that encodes a polypeptide that modulates an immune cell,optionally a dendritic cell or a T cell, optionally a CD4+ T cell.

In one embodiment, the immune modulatory molecule useful for promotinganti-tumor effect is RANKL. RANKL is a molecule that extends thelifespan of DCs in an autocrine fashion. CD40L which enhances thestimulatory capacity of DCs, is also useful for promoting the anti-tumoreffect of DC and tumor cell vaccines. In addition a number of othercytokines are useful including IL-2, IL-7, IL-15, IL-18, and IL-23. Aperson skilled in the art would recognize that other immune modulatorymolecules, including molecules that promote APC function are suitablefor use in constructs of the present application.

In another embodiment, the immune modulatory cassette comprises apolynucleotide that encodes or directs expression of a molecule thatinhibits IL-12 down modulation, for example inhibits IL-10. In oneembodiment, the molecule is a dominant negative IL-10 polypeptide. Inanother embodiment the molecule is a small molecule inhibitor. Inanother embodiment, the molecule is a siRNA or shRNA molecule thatknocks down IL-10 gene expression.

Safety Components The Cell Surface Protein—Use of Immunotoxin to KillTransduced Cells

In certain embodiments of the invention, a cell surface protein (marker)herein referred to as a detection cassette, such as CD19, CD20 HSA,truncated LNGFR, CD34, CD24 or CD25 is delivered into target cells whichfurther selectively clears these cells in vitro and in vivo byadministering an immunotoxin (antibody conjugated to a toxin) directedagainst the cell surface protein. The term “immunotoxin” as used hereinmeans an antibody or fragment thereof that is cytotoxic and/or anantibody or fragment there of that is fused to a toxic agent.Immunotoxins are described in this application and known in the art, forexample, in US patent application no. 20070059275.

Many immunotoxins are approved for use in humans. In one embodiment theimmunotoxin is a murine anti-Tac (AT) monoclonal antibody19 fused tosaporin (SAP)¹⁰⁰ a toxin that irreversibly damages ribosomes by cleavingadenine molecules from ribosomal RNA.21 The inventors have demonstratedboth in vitro and in vivo that the AT-SAP (ATS) complex specificallytarget and kill retrovirally transduced cells that express huCD25. Useof immunotoxins to kill transduced cells are described in CA applicationVector Encoding Therapeutic Polypeptide and Safety Elements to ClearTransduced Cells, filed Mar. 27, 2007 which is incorporated herein byreference.

Activator Polynucleotides

Other safety components that can be introduced into the vectorconstructs disclosed are described in U.S. application Ser. No.11/559,757, THYMIDYLATE KINASE MUTANTS AND USES THEREOF and U.S.application Ser. No. 12/052,565 which are incorporated herein byreference. In one embodiment, the lentiviral construct further comprisesan activator polynucleotide encoding a polypeptide that converts aprodrug to a drug, optionally a modified tmpk polynucleotide. In oneembodiment, the activator polynucleotide comprises a tmpk polynucleotidewith at least 80% sequence identity to a modified tmpk polynucleotide,optionally the sequences listed below.

The safety facet of suicide gene therapy relies on efficient deliveryand stable, consistent expression of both the therapeutic and the safetycomponent genes.

Expression Cassette Variants and Analogs

In the context of a polypeptide, the term “analog” as used hereinincludes any polypeptide having an amino acid residue sequencesubstantially identical to any of the wild type polypeptides expressedby the expression cassette for example, IL-12 or mutant IL-12, in whichone or more residues have been conservatively substituted with afunctionally similar residue and which displays the ability to activatein the context of IL-12, the IL-12 receptor similar to wild-type IL-12or to IL-12 mutants. Examples of conservative substitutions include thesubstitution of one non-polar (hydrophobic) residue such as alanine,isoleucine, valine, leucine or methionine for another, the substitutionof one polar (hydrophilic) residue for another such as between arginineand lysine, between glutamine and asparagine, between glycine andserine, the substitution of one basic residue such as lysine, arginineor histidine for another, or the substitution of one acidic residue,such as aspartic acid or glutamic acid for another. The phrase“conservative substitution” also includes the use of a chemicallyderivatized residue in place of a non-derivatized residue provided thatsuch polypeptide displays the requisite activity.

In the context of a polypeptide, the term “derivative” as used hereinrefers to a polypeptide having one or more residues chemicallyderivatized by reaction of a functional side group. Such derivatizedmolecules include for example, those molecules in which free aminogroups have been derivatized to form amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups,chloroacetyl groups or formyl groups.

Free carboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Also included as derivatives are those peptides which contain one ormore naturally occurring amino acid derivatives of the twenty standardamino acids. For examples: 4-hydroxyproline may be substituted forproline; 5 hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.Polypeptides of the present invention also include any polypeptidehaving one or more additions and/or deletions or residues relative tothe wild type sequence, so long as the requisite activity is maintained.

The methods of making recombinant proteins are well known in the art andare also described herein.

The nucleic acids described herein can also comprise nucleotide analogsthat may be better suited as therapeutic or experimental reagents.

The nucleic acid can also contain groups such as reporter groups, agroup for improving the pharmacokinetic properties of an nucleic acid.

The nucleic acid molecules may be constructed using chemical synthesisand enzymatic ligation reactions using procedures known in the art.

The nucleic acid molecules of the invention or a fragment thereof, maybe chemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules.

Isolated Virus

The retroviral and lentiviral constructs are in one embodiment, packagedinto viral particles. Methods for preparing virus are known in the artand described herein. In one embodiment, the application provides anisolated virus, optionally a lentivirus comprising the vector construct.

Methods of isolating virus are also known in the art and furtherdescribed herein.

Methods of Expressing IL-12 in Cells and Cell Isolation

In one aspect, methods for expressing IL-12 in cells at or above athreshold level are provided. Accordingly in one aspect, the applicationprovides a method of expressing IL-12 in a cell above a threshold level.

The polynucleotides may be incorporated into an appropriate expressionvector which ensures good expression of the IL-12 and/or otherexpression cassettes herein described. For example, vectors describedherein are suitable.

Possible expression vectors include but are not limited to cosmids,plasmids, or modified viruses (e.g. replication defective retroviruses,adenoviruses and adeno-associated viruses), so long as the vector iscompatible with the host cell used. The expression vectors are “suitablefor transformation of a host cell”, which means that the expressionvectors contain a nucleic acid molecule and regulatory sequencesselected on the basis of the host cells to be used for expression, whichis operatively linked to the nucleic acid molecule. Operatively linkedor operably linked is intended to mean that the nucleic acid is linkedto regulatory sequences in a manner which allows expression of thenucleic acid.

The application therefore includes a recombinant expression vectorcontaining a nucleic acid molecule disclosed herein, or a fragmentthereof, and the necessary regulatory sequences for the transcriptionand translation of the inserted protein-sequence.

Suitable regulatory sequences may be derived from a variety of sources,including bacterial, fungal, viral, mammalian, or insect genes (Forexample, see the regulatory sequences described in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)). Selection of appropriate regulatory sequences isdependent on the host cell chosen as discussed below, and may be readilyaccomplished by one of ordinary skill in the art. Examples of suchregulatory sequences include: a transcriptional promoter and enhancer orRNA polymerase binding sequence, a ribosomal binding sequence, includinga translation initiation signal. Additionally, depending on the hostcell chosen and the vector employed, other sequences, such as an originof replication, additional DNA restriction sites, enhancers, andsequences conferring inducibility of transcription may be incorporatedinto the expression vector.

Recombinant expression vectors can be introduced into host cells toproduce a transformed host cell. The terms “transformed with”,“transfected with”, “transformation” “transduced” and “transfection” areintended to encompass introduction of nucleic acid (e.g. a vector orvector construct) into a cell by one of many possible techniques knownin the art. The phrase “under suitable conditions that permittransduction or transfection of the cell” refers to for example for exvivo culture conditions, such as selecting an appropriate medium, agentconcentrations and contact time lengths which are suitable fortransfecting or transducing the particular host. Suitable conditions areknown in the art and/or described herein. The term “transformed hostcell” or “transduced host cell” as used herein is intended to alsoinclude cells capable of glycosylation that have been transformed with arecombinant expression vector disclosed herein. Prokaryotic cells can betransformed with nucleic acid by, for example, electroporation orcalcium-chloride mediated transformation. For example, nucleic acid canbe introduced into mammalian cells via conventional techniques such ascalcium phosphate or calcium chloride co-precipitation, DEAE-dextranmediated transfection, lipofectin, electroporation or microinjection.Suitable methods for transforming and transfecting host cells can befound in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 3rdEdition, Cold Spring Harbor Laboratory Press, 2001), and otherlaboratory textbooks. Suitable methods for transducing cells are knownin the art and are also described herein.

Vector constructs are introduced into cells that are used for transplantor introduced directly in vivo in mammals, preferably a human. Thevector constructs are typically introduced into cells ex vivo usingmethods known in the art. Methods for introducing vector constructscomprise transfection, infection, electroporation. These methodsoptionally employ liposomes or liposome like compounds. Introduction invivo optionally includes intravenous injection and/or intratumoralinjection. These methods are described more fully elsewhere

In certain embodiments, the cell is contacted with a composition vectorconstruct and/or isolated virus described herein, for example anisolated virus comprising a lentiviral vector and a IL-12 expressioncassette, under conditions that permit transduction or transfection ofthe cell. Methods of transducing cells are well known in the art.

In one embodiment, the method of expressing IL-12 in a cell comprisescontacting the cell with a composition and/or vector construct describedherein, for example comprising a lentiviral vector and an IL-12expression cassette, under conditions that permit transduction ortransfection of the cell.

In other embodiments, the cells are optionally transduced withretroviral constructs that drive expression of IL-12 and/or additionalexpression cassettes described herein. Methods of transducing cells arewell known in the art. Methods of transducing cells with lentiviralvectors are also described herein.

In another embodiment, the method further comprises isolating thetransduced cell or a population of transduced cells.

After transduction or transfection with vector constructs comprising anIL-12 expression cassette, and/or detection cassette polynucleotide,cells expressing these molecules are optionally isolated by a variety ofmeans known in the art. In certain embodiments, the cells are isolatedby cell sorting or flow cytometry using an antibody to the detectioncassette encoded selection marker. Additionally cell sorting is usefulto isolate modified cells where the detection cassette is a fluorescentprotein such as EGFP.

In one embodiment cells are isolated from the transduction ortransfection medium and/or the viral preparation. For example the cellsmay be spun down and/or washed with a buffered saline solution.Accordingly, the cells can comprise a population of cells comprisingtransduced and untransduced cells. In certain embodiments, thepopulation of cells comprises at least 1%, 2-5%, 5-10%, 10-15%, 15-20%,20-25%, 25-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%,95-99% or more than 99% IL-12 transduced or transfected cells.

Cells expressing polynucleotides of the invention are, in an alternateembodiment, isolated using magnetic sorting. Additionally, cells may beisolated by drug selection. In one embodiment, a vector comprising adrug resistance gene and a polynucleotides of the invention isintroduced into cells. Examples of drug resistance genes include, butare not limited to, neomycin resistance gene, blasticidin resistancegene (Bsr), hygromycin resistance gene (Hph), puromycin resistance gene(Pac), Zeocin resistance gene (Sh ble), FHT, bleomycin resistance geneand ampicillin resistance gene. After transduction or transfection,modified cells including the drug resistance gene are selected by addingthe drug that is inactivated by the drug resistance gene. Cellsexpressing the drug resistance gene survive while non-transfected ornon-transduced cells are killed. A person skilled in the art would befamiliar with the methods and reagents required to isolate cellsexpressing the desired polynucleotides.

In a further embodiment, the transduced cells are growth arrested.Several methods can be used to growth arrest cells. In one embodiment,the transfected or transduced cells are growth arrested by irradiation.The term “growth arrested” refers to being inhibited for cell division.A person skilled in the art would recognize that the suitableirradiation dose to growth arrest a cell or population of cells may varyupon the cell type and/or number of cells. In one embodiment, the doseis about 75-150G. In another embodiment, for AML the dose of radiationis about 75G.

Host Cells

The disclosure also provides in one aspect a cell (including for examplean isolated cell in vitro, a cell in vivo, or a cell treated ex vivo andreturned to an in vivo site) expressing and/or secreting IL-12 above athreshold limit. In one embodiment, the cell is transduced with a vectorconstruct, virus or composition described herein.

Cells transfected with a nucleic acid molecule such as a DNA molecule,or transduced with the nucleic acid molecule such as a DNA or RNA virusvector, are optionally used, for example, in bone marrow or cord bloodcell transplants according to techniques known in the art.

Any cell may be used for transduction with the vector constructsdescribed herein to obtain a cell expressing IL-12 above the thresholdlevel. In one embodiment, the cell is a cancer cell. In one embodiment,the cancer cell is a primary cancer cell. In a further embodiment, theprimary cancer cell is derived from a subject. The cancer cell isoptionally an allogenic or autologous cell. The cancer cell to betransduced is optionally derived from, propogated from or cloned from acancer cell obtained from a subject. The cancer cell is in oneembodiment obtained from the subject by biopsy. Alternatively, thecancer cell can be obtained from a blood sample, for example in the caseof a leukemia, where the disease cell type is present in the peripheralblood. Methods for isolating cancer cells from a blood sample are knownin the art and/or described herein.

Any cancer cell that can be transduced or transfected is a suitable hostfor transduction or transfection using a composition or vector constructof the application. In one embodiment the cancer cell is a leukemiacell. In one embodiment the leukemia cell is an acute lymphoblasticleukemia (ALL) cell, a chronic lymphoblastic leukemia (CLL) cell,chronic myeloid leukemia (CML) cell, or acute myeloid leukemia (AML)cell. In certain embodiments, the cancer cell is derived from a cancerthat is characterized by or can exhibit periods of remission. In certainembodiments, the cancer cell is a metastatic cancer cell. In otherembodiments, the cancer cell is a lymphoma, myeloma, tumor of the lung,ovary, prostate, breast, melanoma, colon, bladder, liver, pancreas,thyroid, head or neck cancer cell. The immune system is able to seek outcells residing in nearly all parts of the body and therefore all cancerscould be susceptible to this approach including: leukemias, lymphoma,myelomas, tumors of the lung, ovary, prostate, breast, melanoma, colon,bladder, liver, pancreas, thyroid, head and neck.

Cell lines are optionally transduced or transfected. For example human Tcell leukemia Jurkat T cells, human erythro-leukemic K562 cells, CES1,OCIAML1, OCIAML2, and Raji cells are optionally transduced ortransfected with polynucleotides of the described herein. Raji is aburkitts lymphoma line, OCI AML 1 and 2 are acute meylogenous leukemialines, CES1 is a chronic myelongenous leukemia

A cancer cell expresses tumor associated antigens and introduction ofIL-12 and optionally immune modulatory molecules that augment the immuneresponse when the tumor cell is introduced into the subject asdemonstrated by the inventors. In one embodiment, the tumor cell istransduced with a lentiviral construct comprising an IL-12 cassette andoptionally an immune modulatory cassette, wherein the immune modulatorycassette comprises a polynucleotide that encodes a molecule that inducesDC cells and/or T cells. Cancer cells are attractive vehicles forexpressing IL-12 as the immune response is self limiting. Transducedcancer cells elicit an immune response that leads to the eradication ofthe initiating cell. IL-12 levels are thereby self-limited.

Compositions and vector constructs described herein are usefullyintroduced into any cell type ex vivo. The compositions and vectorconstructs described herein may also be introduced into any cell type invivo.

Threshold Level

The inventors have demonstrated that a minimum number of cancer cellsexpressing at least a threshold amount of IL-12 can induce and/orenhance an immune response in a subject. The immune response in someembodiments, leads to loss of non-transduced cancer cells.

In one embodiment, the threshold level is at level is at least 1500pg/mL/10⁶ cells/2 hrs of IL-12. In another embodiment, the thresholdlevel is at least 1500-2500 pg/mL/10⁶ cells/2 hrs, 2500-5000 pg/mL/10⁶cells/2 hrs, 5000-7500 pg/mL/10⁶ cells/2 hrs, 7500-10000 pg/mL/10⁶cells/2 hrs, 10000-12500 pg/mL/10⁶ cells/2 hrs, 12500-15000 pg/mL/10⁶cells/2 hrs, 15000-17500 pg/mL/10⁶ cells/2 hrs, 17500-20000 pg/mL/10⁶cells/2 hrs or at least 20000 pg/mL/10⁶ cells/2 hrs of IL-12.

In another embodiment, a population of cells comprises transduced cellsthat secrete at least about 1500-2500 pg/mL/10⁶ cells/2 hrs, 2500-5000pg/mL/10⁶ cells/2 hrs, 5000-7500 pg/mL/10⁶ cells/2 hrs, 7500-10000pg/mL/10⁶ cells/2 hrs, 10000-12500 pg/mL/10⁶ cells/2 hrs, 12500-15000pg/mL/10⁶ cells/2 hrs, 15000-17500 pg/mL/10⁶ cells/2 hrs, 17500-20000pg/mL/10⁶ cells/2 hrs or at least 20000 pg/mL/10⁶ cells/2 hrs of IL-12.In other embodiments, the population of cells comprise transduced cellsthat secrete at least about 20,000-40,000 pg/mL/10⁶ cells/2 hrs ofIL-12. A person skilled in the art would understand that each cell wouldsecrete varying amounts of IL-12. The population may include cellssecreting less or more than the numbers herein listed or a giventhreshold. The transduced cells as a whole comprise a sufficient numberof IL-12 secreting cells, secreting IL-12 above the threshold level suchthat DC are activated.

The population of cells can comprise transduced and non-transducedand/or transfected and non-transfected cells. In one embodiment, atleast 0.5%. 1%, 2-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-40%,40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-99% or more than 99%of cells in the population of cells are transduced or transfected and/orexpress IL-12.

In a preferred embodiment, the population of cells comprises 1%transduced cells secreting 20,000 pg/10⁶ cells/2 hrs.

The level of IL-12 expression can be determined by a number of methodsincluding methods known in the art and methods described herein.

For example IL-12 levels can be determined by ELISA, cytokine beadassay, intracellular staining, HPLC and MS/MS, or ELISPOT.

Compositions

The application describes compositions comprising an IL-12 expressioncassette and a lentiviral vector as described herein. The vector is forproviding a coding nucleic acid molecule (eg. the expression cassette)to a subject such that expression of the molecule in the cells providesthe biological activity of the polypeptide encoded by the coding nucleicacid molecule to those cells. A coding nucleic acid as used herein meansa nucleic acid or polynucleotide that comprises nucleotides whichspecify the amino acid sequence, or a portion thereof, of thecorresponding protein. A coding sequence may comprise a start codonand/or a termination sequence.

In other embodiments, the composition comprises cells modified with thevector constructs described herein. Such modified cells can beadministered intravenously using methods known in the art i.p., i.v.,intratumorally, stereotactic injections to a variety of sites, directinjections, intramuscularly, etc.

Pharmaceutical Compositions

The pharmaceutical compositions of this invention used to treat patientshaving diseases, disorders or abnormal physical states could include anacceptable carrier, auxiliary or excipient.

The pharmaceutical compositions are optionally administered by ex vivoand in vivo methods such as electroporation, DNA microinjection,liposome DNA delivery, and virus vectors that have RNA or DNA genomesincluding retrovirus vectors, lentivirus vectors, Adenovirus vectors andAdeno-associated virus (AAV) vectors, Semliki Forest Virus. Derivativesor hybrids of these vectors are also useful.

Dosages to be administered depend on patient needs, on the desiredeffect and on the chosen route of administration. The expressioncassettes are optionally introduced into the cells or their precursorsusing ex vivo or in vivo delivery vehicles such as liposomes or DNA orRNA virus vectors. They are also optionally introduced into these cellsusing physical techniques such as microinjection or chemical methodssuch as coprecipitation.

The pharmaceutical compositions are typically prepared by known methodsfor the preparation of pharmaceutically acceptable compositions whichare administered to patients, and such that an effective quantity of thenucleic acid molecule is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA).

On this basis, the pharmaceutical compositions could include an activecompound or substance, such as a nucleic acid molecule, in associationwith one or more pharmaceutically acceptable vehicles or diluents, andcontained in buffered solutions with a suitable pH and isoosmotic withthe physiological fluids. The methods of combining the expressioncassettes with the vehicles or combining them with diluents is wellknown to those skilled in the art. The composition could include atargeting agent for the transport of the active compound to specifiedsites within cells.

Methods of Inducing/Enhancing Immune Responses and Methods of Treatments

The methods disclosed herein are useful for inducing and enhancing animmune response in a subject. In one embodiment, the subject has cancer.In another embodiment, the subject is in remission. In a furtherembodiment, the subject has an increased risk of cancer.

In one embodiment, the application provides a method of inducing orenhancing an immune response in a subject comprising administering atransduced cell or population of cells described herein or a compositioncomprising said cells.

In another embodiment, the application provides a method of inducing orenhancing a memory immune response in a subject.

In one embodiment, the immune response induced or enhanced is a CD4+ Tcell mediated immune response.

The application also provides a method of delivering IL-12 to a subjectfor enhancing cancer treatment comprising:

-   -   generating an IL-12 secreting cell wherein IL-12 secreted per        cell is above a threshold level; and    -   introducing an effective number of the generated IL-12 secreting        cells to the subject.

In another embodiment, the application provides a method of ofsustaining IFNgamma levels induced by IL-12 in a host comprising:

-   -   generating an IL-12 secreting cell wherein IL-12 secreted per        cell is above a threshold level; and    -   introducing an effective number of the generated IL-12 secreting        cells to the subject.

In one embodiment, transduced cells, a population of cells and/or acomposition comprising said cells are administered to a subject Inanother embodiment, the cells, population of cells and/or compositionare administered with an adjuvant. For example, in one embodimentincomplete Freund's adjuvant is used. In addition, the cells, populationof cells and/or composition is administered once, or repeated. Forexample, the cells and or population of cells are administered a secondtime to boost the immune response and/or increase the amount of IL-12delivered or IFNgamma sustained.

In one embodiment, cancer cells are obtained from a subject, andgenetically modified to express and/or secrete IL-12 above a thresholdlevel. The transduced cells or population of cells comprising transducedcells is irradiated and administered to the subject. Accordingly incertain embodiments, clinical use of the modified cells is restricted tothe subject from whom the cancer cell was derived.

Wherein cells additionally express an activator polynucleotide encodinga polypeptide that concerts a prodrug to a drug, for example a modifiedtmpk polynucleotide, cells are optionally not irradiated. Any unwantedcells can be killed upon administration of the prodrug. For example, insome cases, irradiation may negatively effect the ability of thetransduced cells to induce an immune response eg irradiation may causecell death in certain cell populations. Use of an activatorpolynucleotide or other mechanism to remove unwanted cells transplantedinto the subject is alternatively used in such situations.

The methods disclosed herein are useful for treating a variety ofcancers. The inventors have shown that leukemias of a variety of typesare amenable to IL-12 treatment.

Residual disease which can lay dormant during remissions may be targetedby the method disclosed herein. The delayed disease progression of manyleukemias provides a critical window of opportunity for immune-basedapproaches. The present immunotherapy may also rid quiescent cells suchas cancer initiating “stem” cells because it does not requirebiochemically or genetically active targets. Further the presentimmunotherapy may also lead to eradicating metastatic disease.

The methods described herein are also useful to treat solid cancers. Forexample the methods may be used to treat melanoma, renal cancer andprostate cancer.

The cells may be introduced by a variety of routes as disclosedelsewhere including intraperitoneal injection or intravenous infusion.Alternatively, a vector construct, isolated virus or compositioncomprising said construct or virus can be injected intratumorally suchthat transduction takes place in vivo

The number of cells injected or administered is in one embodiment aneffective number to induce an immune response. An immune response can bedetected using a number of methods known in the art including detectinghost T cell recognition of tumor cells in vitro. Alternatively, animmune response can be detected by assessing cytokine profile changes.For example increased expression of IFN-gamma is indicative of an immuneresponse.

In certain embodiments, the methods further comprise monitoring cancerprogression. Cancer progression can be monitored using known methods.

In one embodiment, compositions and vectors of the invention are used totreat cancer by adoptive therapy. In one embodiment, cytotoxiclymphocyte cells are expanded using LV-IL-12 transduced cells in vitro.Adoptive therapy or adoptive (immuno)therapy refers to the passivetransfer of immunologically competent tumor-reactive cells into thetumor-bearing host to, directly or indirectly, mediate tumor regression.The feasibility of adoptive (immuno)therapy of cancer is based on twofundamental observations. The first of these observations is that tumorcells express unique antigens that can elicit an immune response withinthe syngeneic (genetically identical or similar especially with respectto antigens or immunological reactions) host. The other is that theimmune rejection of established tumors can be mediated by the adoptivetransfer of appropriately sensitized lymphoid cells. Clinicalapplications include transfer of peripheral blood stem cells followingnonmyeloablative chemotherapy with or without radiation in patients withlymphomas, leukemias, and solid tumors.

In one aspect of the present invention, donor T cells or stem cells(either embryonic or of later ontogeny) are transduced with vectors ofthe invention. Cells expressing these vectors are isolated andadoptively transferred to a host in need of treatment. In one embodimentthe bone marrow of the recipient is T-cell depleted. Methods of adoptiveT-cell transfer are known in the art (J Translational Medicine, 20053(17): doi; 0.1186/1479-5876-3-17, Adoptive T cell therapy: Addressingchallenges in cancer immunotherapy. Cassian Yee). This method is used totreat solid tumors and does not require targeting the vector-transducedexpressing T-cells to the tumor since the modified T-cells willrecognize the different MHC class molecules present in the recipienthost resulting in cytotoxic killing of tumor cells.

In one embodiment, autologus DC and T cells are contacted ex vivo withIL-12 transduced cancer cells and/or expanded ex vivo and administeredto a subject in need thereor with or without LV-IL-12 secreting cells.

The compositions and vectors are also useful for the reduction of cellproliferation, for example for treatment of cancer. The presentdisclosure also provides methods of using compositions and vectors ofthe disclosure for expressing IL-12 for the reduction of cellproliferation, for example for treatment of cancer.

The application also provides a method of reducing the number of tumorcells or cancer burden in a subject with cancer, or having an increasedlikelihood of developing cancer comprising administering a transducedcell, population of cells, or a composition comprising said cells to thesubject.

In another embodiment, the application provides a method of treating asubject with cancer or an increased risk of developing cancer comprisingadministering a transduced cell, population of cells, or a compositioncomprising said cells to the subject.

Vector constructs containing the nucleic acid molecules of thedisclosure and isolated viruses are typically administered to mammals,preferably humans, using techniques described below. The polypeptidesproduced from the nucleic acid molecules are also optionallyadministered to mammals, preferably humans. The invention relates to amethod of medical treatment of a mammal in need thereof, preferably ahuman, by administering to the mammal a vector construct describedherein or a cell containing the vector construct.

One aspect relates to methods for providing a coding nucleic acidmolecule to the cells of an individual such that expression of thecoding nucleic acid molecule in the cells provides the biologicalactivity or phenotype of the polypeptide encoded by the coding nucleicacid molecule. The method also relates to a method for providing anindividual having a disease, disorder or abnormal physical state with abiologically active polypeptide by administering a nucleic acid moleculeof the present invention. The method may be performed ex vivo or invivo. Gene therapy methods and compositions are demonstrated, forexample, in U.S. Pat. Nos. 5,869,040, 5,639,642, 5,928,214, 5,911,983,5,830,880, 5,910,488, 5,854,019, 5,672,344, 5,645,829, 5,741,486,5,656,465, 5,547,932, 5,529,774, 5,436,146, 5,399,346 and 5,670,488,5,240,846. The amount of polypeptide will vary with the subject's needs.

The optimal dosage of vector may be readily determined using empiricaltechniques, for example by escalating doses (see U.S. Pat. No. 5,910,488for an example of escalating doses).

The method also relates to a method for producing a stock of recombinantvirus by producing virus suitable for gene therapy comprising modifiedDNA encoding a gene of interest. This method preferably involvestransfecting cells permissive for virus replication (the viruscontaining therapeutic gene) and collecting the virus produced.

Cotransfection (DNA and marker on separate molecules) may be employed(see eg U.S. Pat. No. 5,928,914 and U.S. Pat. No. 5,817,492). As well, adetection cassette or marker (such as Green Fluorescent Protein markeror a derivative) may be used within the vector itself (preferably aviral vector).

Combination Treatments

In certain embodiments, the vector constructs, transduced cells,population of cells and or compositions comprising these, areadministered in combination with other therapies. For example, the thevector constructs, transduced cells, population of cells and orcompositions comprising these may be administered before or afterchemotherapy suitable for the cancer being treated. In other embodimentswherein the cancer is a solid cancer, the vector constructs, transducedcells, population of cells and or compositions comprising these areadministered before or after surgery.

In one embodiment, cancer cells are harvested from a subject's bloodbefore the combination treatment, optionally chemotherapy, is started.The cancer cells are then transduced with a LV-IL-12. Transduced cellsare frozen for later use and administered when the subject is inremission.

Dosing

The methods provide in certain embodiments, that a composition,transduced cell, population or cells, or vector construct describedherein is administered to the subject. The compositions, cells or vectorconstructs of the present application may be administered at least oncea week in one embodiment. However, in another embodiment, thecomposition, transduced cell, population or cells, or vector constructmay be administered to the subject from about one time per week, onetime per 14 days, or 28 days. The administration may be repeated 1, 2,3, 4, 5, 6 or more times. In another embodiment, administration is aboutonce daily for a given treatment, for example for rIL-12 therapy. Thelength of the treatment period depends on a variety of factors, such asthe severity of the disease, the age of the patient, the concentrationand the activity of the compounds of the present application, or acombination thereof. In one embodiment, the treatment is chronictreatment and the length of treatment is 1-2 weeks, 2-4 weeks or morethan 4 weeks. The treatment regimen can include repeated treatmentschedules. It will also be appreciated that the effective amount ordosage of the compound used for the treatment or prophylaxis mayincrease or decrease over the course of a particular treatment orprophylaxis regime. Changes in dosage may result and become apparent bystandard diagnostic assays known in the art. In some instances, chronicadministration may be required.

The number of cells administered varies with the expression level of thetransduced cell or population of cells. For example, where the IL-12expressing cells express over 20000 pg/mL/10⁶ cells/2 hrs IL-12, as fewas 5000 or 0.5% of a population of cells comprising IL-12 expressingcells may be sufficient for the methods described herein. However wherethe IL-12 expressing cells express only 2000 pg/mL/10⁶ cells/2 hrsIL-12, greater than 100000 or 10% of a population of cells comprisingIL-12 expressing cells may be needed.

In one embodiment, 1-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70,70-80, 80-90, 90-100 or more than 100×106 cells are administered. Inanother embodiment, 10⁶-10⁹ cells are administered. Where the cellsproduce greater than 2000 pg/ml/10⁶ cells/2 hrs greater than 10% of thepopulation of cells express IL-12. Wherein the cells express 20, 000pg/ml/10⁶ cells/2 hrs, at least 0.5% of the population of cells expressIL-12.

Polypeptide Production and Research Tools

A cell line (either an immortalized cell culture or a stem cell culture)transfected or transduced with a polynucleotide of the invention (orvariants) is useful as a research tool to measure levels of expressionof the coding nucleic acid molecule and the activity of the polypeptideencoded by the coding nucleic acid molecule.

The invention includes a method for producing a recombinant host cellcapable of expressing a nucleic acid molecule of the inventioncomprising introducing into the host cell a vector of the invention.

The invention also includes a method for expressing a polypeptide in ahost cell of the invention including culturing the host cell underconditions suitable for coding nucleic acid molecule expression. Themethod typically provides the phenotype of the polypeptide to the cell.

Another aspect of the invention is an isolated polypeptide produced froma nucleic acid molecule or vector of the invention according to a methodof the invention.

Another aspect relates to a system or model for testing the mechanism ofIL-12 mediated rejection of cancer. In one embodiment the system is anin vitro system. Understanding the underlying mechanism that leads to aneffective anti-leukemia immune response is greatly facilitated byestablishing in vitro assays which mimic in vivo observations. This isuseful for comparing and adapting murine models to human disease. In oneembodiment, the in vitro system comprises murine bone marrow derived DCs(grown for 6-9 days in GM-CSF) induced to mature (increased expressionof CD80) in the presence of both spleen cells+70Z/3-IL-12 producingcells (but not with either alone). Maturation does not occur ifnon-transduced 70Z/3 cells are substituted for the 70Z/3-IL-12 cells.Selected populations from the spleen are added and/or removed (immatureT cells, CD4⁺ T cells, CD8⁺ T cells, NKT cells, NK cells, DC precursors)to define the critical cell types that are required for 70Z/3-IL-12mediated DC maturation.

In one embodiment the system comprises human leukemia cells expressingIL-12 and/or a mouse model susceptible to developing cancer to determinethe mechanism by which Interleukin-12 (IL-12) provokes an immuneresponse which, in mice, results in complete rejection of leukemia. Inone embodiment, the system permits analysis of the interactions of Tcells, dendritic cells (DC), leukemia cells and the cytokines that theyproduce in established murine in vitro and in vivo systems. In anotherembodiment, the system permits optimization of the parameters essentialfor engineering primary samples of human leukemia cells to expressquantities of IL-12 above necessary thresholds established in the murinesystem. In a further embodiment, the system is useful to establish invitro conditions to determine how primary human leukemia cellsexpressing IL-12 interact with the autologous DCs and T cells.

EXAMPLES Example 1

Direct injection of recombinant IL-12 has shown effectiveness in somemouse models of leukemia.[9-13] while initial human trials employingthis approach were less promising ([14-17] and discussed in [4]). It iswell recognized in the literature that IL-12-induced anti-leukemiaactivity is largely mediated by the secondary secretion of IFN-γ.[13]Gollob et al., in particular, have suggested that the induction andmaintenance of IL-12-induced IFN-γ was a key component of effectivetherapy in patients with metastatic renal cell cancer.[18] However theconcomitant induction of antagonistic effects with elevated IFN-γ levelscontinues to pose a challenge and is the impetus for a number of groupsto continue testing the efficacy of recombinant IL-12 followingdifferent dose and time protocols[7, 8, 19-21] and to evaluate thetherapeutic potential of cell-based IL-12 gene therapy ([22-27] anddiscussed in [4, 13]) in order to overcome this.

More recent clinical trials have included approaches such asintraleukemial injection of IL-12 secreting fibroblasts and dendriticcells, methods that have proven effective in mouse models. To date,these approaches have not had a significant impact on patientsurvival[15-17]. Finding the reason for this disconnect is of paramountimportance.

The inventors recently published a model of ALL in which one variant ofthe 70Z/3 murine pre-B cell leukemia line, 70Z3-L, is lethal insyngeneic mice while another variant, 70Z/3-NL, elicits a protectiveimmune response (27). The 70Z/3-L cells, although unable to initiateimmunity, were readily rejected when an immune response was firstinitiated against 70Z/3-NL cells. Therefore, our model is amenable totesting whether IL-12 can initiate a specific immune response,recognition of 70Z/3-L and survival of challenged animals. 70Z/3leukemia is reminiscent of human ALL with neoplastic lesions arising inthe liver, spleen, lymph nodes, bone marrow and rarely the centralnervous system. Among the most common physical manifestations of thedisease are ascites and splenomegaly.

Materials and Methods Animals.

Female (C57BI/6×DBA/2)F1 mice (referred to as BDF1), 8-12 weeks, oldwere purchased from the Jackson Laboratories (Bar Harbor, Ma). Mice werekept under sterile conditions in the specific pathogen free (SPF) animalfacility at the Ontario Cancer Institute, Princess Margaret Hospital,Toronto, Ontario, Canada. Mice are fed an irradiated diet and autoclavedtap water. Animals are terminated by CO₂ asphyxiation and cervicaldislocation. The Animal Care Committee of the Ontario Cancer Instituteapproved all experimental protocols employed.

Tumor Cells.

Leukemia Cells.

70Z/3-L leukemia cells (described in[28]), derived from BDF₁ mice, weremaintained in IMDM with 5% heat inactivated fetal bovine serum (HYCLONE,South Logan, Utah, USA), 100 μg/mL penicillin-streptomycin or 100 μg/mLkanamycin (GIBCO-Invitrogen), and 5.5×10⁻⁵ M β-mercaptoethanol (referredto as complete IMDM) in a humidified atmosphere at 37° C. and 5% CO₂.Cell concentrations were kept at 5-10×10⁵ cells/mL.

Lentiviral Vector Construction.

Lentiviral vectors expressing IL-12 cDNA were constructed by a methodsimilar to that described by Yoshimitsu et al.[29] with modification.Plasmid pORF-mIL12 (IL-12elasti(p35::p40) Mouse (p35::p40)) (InvivoGen,San Diego, Calif.) was modified by creating EcoRI and BamHI restrictionenzymes sites, upstream and downstream of the IL-12 gene respectivelyusing a QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla,Calif.). This resulting construct was then digested with EcoRI/BamHI(New England Biolabs). Murine IL-12 cDNA was purified afterelectrophoresis on a 1% agarose gel, and then subcloned into the pHR′ LVbackbone downstream of the elongation factor 1 alpha (EF1α) promoter.Positive plasmid clones for pHR-cPPT-EF1α-muIL-12-WPRE (i.e. LV-muIL-12)were identified by diagnostic restriction enzyme digestion analyses andsubsequent DNA sequencing (Innobiotech, Toronto, ON, Canada).

Viral Production and Transduction of the Cells.

Concentrated LVs were produced by a transient triple-transfection methodusing pHR-cPPT-EF1α-muIL-12-WPRE and accessory plasmids onto 293Tmonolayers by calcium phosphate.[30, 31] An approximate vector titre wasestimated based on LV/enGFP[29] production and testing on naïve 293Tcells that occurred in parallel. The murine pre-B leukemic cell line,70Z3-L, was then transduced with an approximate multiplicity ofinfection (MOI) of 20. Single cell clones, obtained by limiting dilutionin 96 well plates at population densities of less than 0.3 cells/well,were then quantitated for IL-12 production/10 cells/mL/2 hrs using acommercially available IL-12 ELISA kit (BD Biosciences, San Jose,Calif.).

In Vivo Challenge Experiments.

In Vivo Challenge Experiments.

Leukemia cells and transduced cells were grown in complete IMDM and werewashed 3 times with 30 mL of phosphate buffered saline (PBS) with Ca²⁺and Mg²⁺. The cells were resuspended at 5-10×10⁶ cells/mL in PBS andinjected into the animals in a volume of 100-200 μL. Mice received IPinjections that were performed on the right side of the abdomen using a1 mL syringe with a 26-gauge needle.

Serum Collection.

Serum collection in live mice was achieved by puncturing the saphenousvein with a sterile needle and collecting the blood in a serum separatortube (BD, NJ, USA). These tubes were centrifuged at 10,000 RPM for 5minutes, the serum was then transferred to a micro centrifuge tube andfrozen at −20° C. until use.

Intraperitoneal Administration of rIL-12.

Recombinant mouse IL-12 was purchased from R&D Systems, Minneapolis,USA. Mice were injected IP with 10⁶ 70Z/3-L cells in 100-200 μL PBS onday 0 followed by daily injections of 0.1-20 ng/mouse/day rIL-12 in PBSfor a period of 14 days. A secondary challenge consisted of IP injectionof 10⁶ 70Z/3-L cells 70 days after primary challenge, carried out in themanner just described. For the delayed rIL-12 treatments mice receivedan IP injection of 10⁴ 70Z/3-L cells in 100-200 μL PBS on day 0.Thereafter groups of 4 or 5 mice received 14 successive rIL-12 IPinjections of 20 ng/mouse/day but the initiation of these injections wasdelayed by between 0 and 5 days. The animals were monitored daily forthe appearance of symptoms both during the injection period andfollowing the end of the injections.

Intraperitoneal Administration of Leukemia Cell-Produced IL-12.

Interleukin-12 secreting cells were produced as described above. Micewere injected IP with 10⁶ transduced cells or a mixture of transducedand naïve cells in various proportions in 100-200 μL PBS. A secondarychallenge consisted of IP injection of 10⁶ 70Z/3-L cells or 10⁶ L1210cells more than 110 days after primary challenge carried out in themanner just described. The animals were monitored daily for theappearance of symptoms following injection.

Challenge in-Depleted Animals.

Mice were depleted of CD4⁺, CD8⁺ or both T cell subsets as well as NKcells and IFN-γ using specific antibodies. The hybridoma GK1.5 isdirected against CD4⁺ T cells, YTS169 against CD8⁺ T cells, HB170(R4-6A2) against IFN-γ and the hybridoma HB9419 was used to produce anisotype control antibody. All hybridomas were obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va., USA). The lines weregrown in 2.5 litres of complete IMDM or OptiMEM in Lifecell culture bags(Lifecell Tissue Culture, Baxter Corporation, Concord, Ontario, Canada)in a humidified atmosphere at 37° C. and 5% CO₂ until a live cell count(using trypan blue exclusion) revealed 30% dead cells in the culture.The media was then centrifuged and filtered to remove cells and cellulardebris. The antibodies were purified from the media using an affinitycolumn of packed sepharose beads (Gammabind G, Amersham BiosciencesCorp, Piscataway, N.J., USA) and concentrated with Centriprep YM-30columns (Millipore, Billerica, Mass., USA) before dialysis in PBS. NKcells were depleted using an anti-asialoGM1 antibody produced by WakoBioproducts (Richmond, Va.). Rabbit IgG (Sigma-Aldrich) was used as acontrol for the anti-asialoGM1 antibody. The T cell subset and IFN-γantibodies were injected on days −1, 3, 7, 10 and 14. The doses usedwere 1 mg of antibody on day −1 and 500 μg for the remaining injections.The NK cell depleting antibody was injected on days −1, 4, 9 and 14using the recommended dilution.[32] Control isotype antibodies wereinjected following the same dose and schedule as their correspondingdepleting antibodies. The depletion potential of the antibodies wasdemonstrated in vivo prior to their use in our experiments by injectingmice with a range of concentrations and subsequently examining tissuesby flow cytometry to quantify cellular subsets, or examining serum forthe presence of cytokines by ELISA. This experiment was conducted twiceto test both model systems. In each case, cells were injected on day 0:either 10⁶ 70Z/3-L cells followed by 14 daily injections of rIL-12 (10or 20 ng/mouse/day) or 10⁶ 70Z/3-L vector-transduced cells of the LV12.2clonal line. Controls included mice injected with 70Z/3-L alone and miceinjected with PBS alone according to the appropriate injection schedule.

Bead Assay for Cytokine Levels in the Serum.

Mice were injected IP on day 0 with 10⁶ 70Z/3-L cells in 100 μL PBS andtreated daily with 100 μL preparations of PBS alone or containing lowdoses of rIL-12 (10 or 20 ng/mouse/day) for 14 days. Control groupsincluded mice injected daily for 14 days with PBS in the absence of70Z/3-L cells and rIL-12, and a group that was left entirely untreated.Alternatively, for the leukemia cell-mediated IL-12 therapy experiment,mice were injected on day 0 with 10⁶ 70Z/3-L cells in 100 μL PBScontaining various proportions of the 70Z/3-L vector-transduced cellline LV12.2 (0.5%, 1% and 10%). Control groups included mice injectedwith 70Z/3-L cells alone or PBS alone. Serum was non-terminallycollected from all groups on days 7, 10 and 20 before their dailyinjection by puncture of the saphenous vein as described above. All micefrom the group receiving 70Z/3-L cells alone in the leukemiacell-produced IL-12 therapy model had perished by day 20 such that serumwas not collected from this group. Serum samples were diluted 1/5 andstained according to the protocol provided with the Mouse InflammationCytometric Bead Array Kit (BD, San Diego, Calif., USA). Standards wereprepared in triplicate from independent dilutions and flow cytometry wasdone using a FACScan (Becton Dickinson, Oakville, ON). Acquisition wasperformed using CellQuest software version 3.1.

Southern Blot to Determine Gene Copy Number.

The muIL-12 gene copy number of vector-transduced 70Z3-L clones wasdetermined by Southern blot as described before.²⁴ Briefly, 5 μg ofgenomic DNA extracted from vector-transduced or naïve control 70Z3-Lcells was treated with both EcoRI and HindIII (New England Biolabs) andelectrophoresed onto a 0.8% agarose gel. Next the agarose gel was washedand transferred onto a positively charged nylon membrane (Bio-Rad).²⁵ A746 bp fragment containing the WPRE sequence of LV-muIL-12 to be used asthe hybridization probe was amplified by PCR (Forward primer;5′-tgctccttttacgctatgtgg-3′, Reverse primer; 5′-tcgttgggagtgaattagcc-3′)employing the PCR DIG Probe Synthesis kit (Roche). Southernhybridization was performed using the DIG Luminescent Detection kit(Roche), according to the manufacturer's instructions. Serial dilutionsof the LV-muIL-12 plasmid (see above) in mouse genomic DNA were used asWPRE standards. The results were analyzed using NIH image software andpresented as copies/genome.

Results

Intraperitoneal Administration of rIL-12 Protects Mice Challenged with70Z/3-L.

Interleukin-12 is known to be a potent modulator of the immune responseattributed with a number of anti-leukemia effects including, but notlimited to, T cell-mediated antigen-specific leukemia clearance. Thismolecule has been approved for clinical use but optimum deliveryprograms have yet to be defined. In an attempt to alter the course of70Z/3-L leukemia, we began by testing the effect of IP administration ofrIL-12 on the appearance of morbidity after IP injection of 10⁶ 70Z/3-Lcells. Doses of 0.1-20 ng/mouse/day for 14 days, which are at least20-fold below the maximum tolerated dose in mice were tested. In FIG. 1a, the inventors show that doses above 10 ng were sufficient tosignificantly improve the survival of animals (p=0.002).

Intraperitoneal Administration of rIL-12 Leads to Long-Term ProtectiveImmunity Against the 70Z/3-L Leukemia.

Next addressed is whether the results observed above were due solely tothe acute effects of IP administered rIL-12 on innate responses or tothe induction of a long-term adaptive immune response in the mice. Toaccomplish this, mice received IP injections of 10⁶ 70Z/3-L cells, weretreated for 14 days with 20 ng/mouse/day rIL-12, subsequently challenged70 days later by IP injection of 10⁶ 70Z/3-L cells and monitored for theappearance of symptoms. A group of naïve mice was included to controlfor the efficiency of the cells to cause disease. FIG. 1 bii shows thatall animals first treated with IP administration of rIL-12 (FIG. 1 bi)survived a secondary challenge with 70Z/3-L cells in the absence offurther IL-12 therapy. Thus, IP administration of rIL-12 not onlyprotected against the primary 70Z/3-L challenge but also establishedlong-term protective immune memory.

Intraperitoneal Administration of rIL-12 Protects Animals withPre-Established 70Z/3-L Leukemia.

To determine if IP administration of rIL-12 can lead to leukemiaclearance as well as protection from a developing neoplasm, treatmentinitiation was delayed to allow for dissemination of the disease. Theseexperiments were conducted starting with 10⁴ 70Z/3-L cells injected IPbecause of their rapid growth. This dose is still lethal to 100% of micein approximately 20 days. Initiation of rIL-12 administration wasdelayed by 0 to 5 days and continued for 14 days following the firstinjection. We found that the initiation of rIL-12 therapy could bedelayed by 5 days and still achieve significant protection against theleukemia (FIG. 1 c). The differences between the survival curves of thesix treatment groups are not statistically significant and longer delayswere not tested.

CD4⁺ and CD8⁺ T Cells are Required for the rIL-12-Mediated Rejection of70Z/3-L Cells after IP Administration.

Depleting antibodies were used to determine which cell types mediate therIL-12-induced rejection of 70Z/3-L leukemia after IP administration.FIG. 1 d shows that both CD4⁺ and CD8⁺ T cells are important asdepletion of either population eliminates immune protection in allanimals. The mean survival was 14 days for mice depleted of CD8⁺ Tcells, 23 days for mice depleted of CD4⁺ T cells and 13 days for micedepleted of both T cell subsets. The three curves are not statisticallydifferent from each other. Neutralizing antibodies against IFN-γ wereincluded to examine its role in the rejection response. This abrogatedthe protective effects of IP administered rIL-12 demonstrating thatIFN-γ plays an essential role in leukemia rejection. Although theimportance of NK cells has been shown in other models of IL-12therapy[33, 34] changes in rejection of the 70Z/3-L leukemia were notobserved when NK cells were depleted in this treatment modality (FIG. 1d).

Generation of IL-12 Secreting Leukemia Cells by Implementation ofLentiviral Transduction.

In light of these results, to the option of developing a leukemiacell-mediated approach for the delivery of IL-12 treatment was explored.FIG. 2 a shows the lentiviral construct with an IL-12 fusion transgeneunder control of the EF-1α promoter that was generated. Aftertransducing 70Z3-L cells with an approximate MOI of 20, single cellclones were derived as described in Materials and Methods. Supernatantsfrom these clonal cell lines were tested for the production of IL-12.The range of secretion from selected clones varied from approximately250 to 91,000 pg/mL/10⁶ cells/2 hrs and these levels remained stableover time as shown in FIG. 2 b. Furthermore, the different levels ofIL-12 measured did not seem dependent on cell growth kinetics, nor onsurvival, as the in vitro growth properties of the vector-transducedclones were similar as measured by thymidine incorporation and visualinspection. Southern Blot analysis demonstrated that no clone had morethan 7 proviral integration events.

Only a Small Proportion of Vector-Transduced 70Z/3-L Cells ProducingIL-12 are Required to Confer Immunity.

Whether the production of IL-12 by vector-transduced 70Z/3-L cells wouldelicit a protective immune response was determined by injecting 10⁶cells of each of 12 clones, spanning a range of secretion levels, intothe abdominal cavity of BDF₁ mice. The three lowest producing clones(range: 200-1,000 pg/mL/10⁶ cells/2 hrs) failed to elicit an immuneresponse and mice injected with these cells progressed towards death. Incontrast, all mice injected with 10⁶ cells of the ten highest producingclones (range: from 1500-40000 pg/mL/10⁶ cells/2 hrs) survived (FIG. 3).To date, the majority of the mice included in this study have survivedpast 2 years post-injection.

One 70Z/3-L transduced clone, LV12.1 which produces approximately 21,500pg/mL/10⁶ cells/2 hrs, was mixed with naïve 70Z/3-L cells to determineif the inclusion of IL-12 producing vector-transduced cells would resultin the elimination of non-producing cells also. As little as 2% of thevector-transduced cells were sufficient to confer complete protection(FIG. 4 a). To further examine the efficacy of producer/non-producerproportions, two other 70Z/3-L transduced clones were selected thatdiffered in IL-12 production by 10-fold (clone LV12.3: 2,000 pg/mL/10⁶cells/2 hrs vs. clone LV12.2: 20,000 pg/mL/10⁶ cells/2 hrs). In thiscase, as few as 0.5% (i.e. 5,000 LV12.2 cells in 10⁶ total cells) of thehigher producing clone was sufficient to confer protection to 80% of themice but 0.1% failed to protect any mice. However, even 10% (i.e.100,000 LV12.3 cells in 10⁶ total cells) of the lower producing clonewas insufficient to protect, indicating that a threshold of IL-12production per vector-transduced cell is required to elicit an effectiveimmune response (FIG. 4 b).

Leukemia Cell-Mediated IL-12 Therapy Leads to Specific Long-TermProtective Immunity Against the 70Z/3-L Leukemia.

More than 110 days post IP injection with 10⁶ LV12.2 cells, mice werechallenged with either 10⁶ cells of the parental leukemia line 70Z/3-Lor another well-characterized B-cell leukemia, L1210, and monitored forthe appearance of symptoms. Groups of naïve mice were included tocontrol for the efficiency of both the 70Z/3 and L1210 cells to causedisease. FIG. 5 shows that all animals to survive the initial insultwith LV12.2 were immune to subsequent challenge with 70Z/3-L but notL1210. Thus, cell-mediated IL-12 therapy leads to specific long-termprotective immunity.

CD4⁺ T Cells are Primarily Required for Leukemia Cell-Mediated Rejectionof 70Z/3-L Cells.

Depleting antibodies were used to determine which cell types mediate theIL-12-induced rejection of 70Z/3 leukemia. FIG. 6 shows that the CD4⁺ Tcell subset is of primary importance unlike in the IP administeredrIL-12 therapy model above. The mean survival of leukemia challengedmice was 37 days for animals depleted of CD4⁺ T cells and 18 days forthose depleted of both T cell subsets. The curves are statisticallydifferent (p=0.003), suggesting an important role for CD8⁺ T cells butonly in the absence of CD4⁺ T cells. The CD8⁺ T cell subset alone is notsufficient to confer protection. Furthermore, the neutralization ofIFN-γ did not diminish the protective effect as was seen with IPadministered rIL-12 therapy (FIG. 6). This was a surprising result andprompted us to further interrogate the regulation of IFN-γ and variousother inflammatory cytokines in each model.

In Vivo Cytokine Regulation.

Interleukin-12 induces the secretion of other cytokines that can haveagonistic, antagonistic or synergistic effects and can influence thespecific immune response that is initiated.[6-8, 18, 35-38] It wastherefore important to measure the regulation of some of these cytokinesin vivo to better understand how leukemia rejection is accomplished andshed some light on the results of our neutralization experiments. Forthis purpose we employed a flow cytometry technique that detects a panelof inflammatory cytokines, including IL-12 p70, TNF-α, IFN-γ, MCP-1,IL-10 and IL-6 in serum. Mice received IP injection of 10⁶ 70Z3-L cellson day 0 and daily IP injections of either 10 or 20 ng rIL-12/mouse/dayfor 14 days. Serum samples were collected on days 7, 10 and 20.Alternatively, mice were challenged with an IP injection of 10⁶ 70Z3-Lcells on day 0 spiked with various proportions (0.5%, 1% and 10%) ofvector-transduced cells and serum samples were collected according tothe same schedule as described above. The results of these two assaysare shown in FIG. 7.

The levels of IL-10 induced on day 20 are significantly higher afterleukemia cell-mediated therapy as compared to IP administered rIL-12therapy (p<0.0017) but are not significantly different between IL-12treated and control groups for either mode of delivery at any timepoint. Likewise, the levels of IFN-γ and TNF-α are significantly higherin response to IL-12 secreted from vector-transduced cells (p<0.0015 and0.0110 respectively). Of note, however, is that leukemia cell-mediatedtreatment groups show significantly higher levels of IFN-γ than thecontrol groups on day 7 (p=0.0007) but resolve to near basal level byday 20.

DISCUSSION

The inventors demonstrate that IP administered low dose rIL-12 therapycan elicit a protective immune response in leukemia-bearing mice andthat an effective approach to deliver IL-12 is via the leukemia cellsthemselves. Remarkably few transduced leukemia cells are needed toachieve protection provided a sufficient amount of IL-12 is produced percell, and that protection is achieved in a manner distinct from thatwith IP administered rIL-12 therapy.

Given the key role that IL-12 plays in the initiation of effectiveimmune responses in various leukemia models, the potential for cytokinetherapy using a murine model of ALL was re-examined. It had previouslybeen found that 70Z/3-L cells lead to the rapid death of mice injectedwith as few as 10² cells. In contrast, variants of this line that arerecognized by the immune system and subsequently rejected wereestablished. Mixing as few as 10 of these non-leukemic variants with 10⁶70Z/3-L cells resulted in complete rejection of all 70Z/3 cells.[39]While why these variants are recognized by the immune system has not yetbeen determined, these experiments revealed that 70Z/3-L cells can berejected if the immune system can be modulated appropriately; makingthis experimental system amenable to the study of IL-12-inducedanti-leukemia activity.

Interleukin-12-based therapies have not become front line cancertreatments in part because studies often report low response rates amongpatients.[6-8] The poor outcomes associated with IL-12 treatment inthese clinical studies can be explained by the physiological response toIL-12-induced IFN-γ. For example high levels of IL-12, and consequentlyIFN-γ, have been shown to induce IL-10 and lead to downmodulation ofIL-12 responsiveness in the host.[6] However, Gollob et al reportchronic T helper type-1-like immune activation involving IFN-γproduction is necessary for rhIL-12-induced antitumor effects.[18]

Previous groups have demonstrated that administration of IL-12 at dosessignificantly below the maximum tolerated dose can avoid the inductionof antagonistic mechanisms.[20] The inventors demonstrated that IPadministration of a dose as low as 10-20 ng of rIL-12 daily for 14 days,equivalent to 500-1,000 ng/kg, is sufficient to significantly increasethe survival of mice injected with 70Z/3-L. This dose is effectiveagainst an established leukemia burden and rejection leads to long-termimmune memory in a T cell-dependent manner.

Other strategies for delivery of IL-12 were investigated 70Z/3-L cellscan be readily transduced with our novel lentiviral construct. Differentvector-transduced clones produce varied amounts of IL-12. This appearsto be a stable trait as we have measured similar levels of secretedIL-12 for each clone on 2-5 independent occasions. The vector copynumber in these clones was determined but this alone does not explainthe variable secretion levels, nor does their rate of proliferation. Onepossible explanation, however, is that the variable secretion is aresult of different integration sites and the effect of different genescontrolling transgene regulation.

The establishment of clones that produce different levels of IL-12 hasallowed examination of the relationship between IL-12 production and theproportion of IL-12⁺ vector-transduced vs. IL-12⁻ naïve 70Z/3-L cellsnecessary for immune activation. To date, this potentially criticalaspect of cell-mediated cytokine therapy has not been thoroughlyexamined. A very small proportion of IL-12 producing vector-transduced70Z/3-L cells are sufficient to trigger a protective immune response.For one clone, LV12.2, 5,000 such vector-transduced cells (but not1,000) were sufficient to save 80% of the mice injected with 10⁶ 70Z/3-Lcells. This result could indicate either that a critical number of“hits” or a sufficient amount of IL-12 is required to trigger an immuneresponse. A reasonable interpretation of “hit” might be an encounterbetween an IL-12 producing vector-transduced 70Z/3-L cell and anappropriate APC, such as a DC. The alternative explanation proposed isthat these 5,000 vector-transduced cells simply deliver a sufficientquantity of IL-12 into the system to trigger an immune response in amore direct fashion. To determine which of these explanations iscorrect, a different clone, LV12.3, that produces 10-fold less IL-12 percell was employed. Titrated numbers of vector-transduced cells wereinjected along with 10⁶ 70Z/3-L naïve cells. Even 100,000 of suchvector-transduced cells failed to confer protection. This representstwenty-fold more cells and twice the potential IL-12 released into thesystem. Together, these results suggest that it is the number of “hits”that matter rather than the absolute amount of IL-12, but that toqualify as a “hit”, the vector-transduced 70Z/3-L cell must produceIL-12 above a certain threshold.

These findings have important implications for clinical trial design andmay explain at least part of the differences observed between murinestudies, in which IL-12 can initiate a curative immune response, andhuman studies, in which the immune response is modest and patientsurvival is normally unaffected. The protocols used in mouse studiesusually involve selection of clones that secrete relatively high levelsof IL-12 and frequently the preparation administered consists of 100%IL-12 secreting cancer cells. In contrast, human studies generally relyon freshly obtained populations of cancer cells that are difficult toclone. Therefore bulk populations of cells are transduced and averageamounts of IL-12 produced by these populations are measured. In casesreported to date, these average amounts are far below what is predictedto be necessary to elicit protective immunity and there is noinformation on the distribution of production levels within thesepopulations.

The IL-12-induced anti-leukemia activity in our two models is Tcell-dependent but the subsets that are critical differ depending on themode of IL-12 delivery. The role of IFN-γ also appeared to differ,prompting us to look at its in vivo regulation along with a number ofother inflammatory cytokines. This was done using a flow cytometry basedcytokine bead assay. The regulation of IL-10, IFN-γ and TNF-α are ofparticular interest in our model systems because IL-10 is known to bethe most biologically relevant antagonist of IL-12,[4] IFN-γ is maymediate the effects of IL-12[4, 13] and a combination of IFN-γ and TNF-αis required for the development of CD4⁺ CTLs.[5]

The fact that IL-10 production was not elevated above background in anyof our treatment groups suggests that the amount of IL-12 administeredwas sufficiently low as to avoid the induction of antagonistic moleculesand dampening of the biologic effect. Measured levels of IFN-γ weresignificantly higher in the treated groups receiving leukemiacell-produced IL-12 as compared to controls on day 7 but were notsignificant by day 10 and returned to near baseline by day 20.Furthermore, IFN-γ production was significantly greater in the leukemiacell-mediated model in general. In light of these results, it isprobable that the leukemia cell-mediated IL-12 therapy neutralizationexperiment did not demonstrate a critical role for IFN-γ simply becausethe neutralizing antibody was overwhelmed by the levels produced. Thereis ample literature describing how IL-12 leads to the increasedmaturation of DCs, the production of IFN-γ and more efficient antigenpresentation by the IFN-γ-dependent up-regulation of MHC-II andco-stimulatory molecule expression. T-helper lymphocytes are driven byIFN-γ to differentiate with a type-1 functional profile and subsequentlypromote the strong CD8+CTL response that we saw with IP administeredrIL-12 therapy. However, there is also a literature describing a rolefor CD4+CTLs in models of infection[5, 40, 41] and more recently intumor immunology[42-46]. It is possible that the IFN-γ and TNF-α richenvironment resulting from leukemia cell-mediated therapy led to thedevelopment of an effector CD4⁺ population. This could account for thedifferential importance of T cell subsets in our two models and explainthe distinct results of the neutralization experiments. The major thrustof tumour vaccination research has traditionally focused on targetingCD8⁺ CTLs, which require stimulation by a CD4⁺ helper T cell population,to affect tumour clearance but the clinical response has been limited.Directly targeting CD4⁺ effector cells may be important to achieve amore robust anti-tumour response.

Despite the beneficial effects of IFN-γ that we have highlighted above,a dampening of the response with repeated administration is still ofconcern in models of IL-12 therapy. An important attribute of ourleukemia cell-mediated model is that a sufficient immune response isinitiated and the leukemia cleared but the signal is self-limitingbecause the source of IL-12 into the system is the cancer cells thatare, themselves, the target of therapy. As the leukemia cells arerejected, the source is reduced and IFN-γ levels return to baselinewithout a significant increase in the antagonistic molecule IL-10.

IL-12, given at doses below the level leading to the induction ofantagonistic mechanisms, is sufficient to launch a protective immuneresponse against 70Z/3-L ALL cells and complete clearance of theleukemia. The mode of IL-12 delivery can have a profound impact on thenature of the immune response that is mounted and demonstrates acritical role for CD4⁺ cells in our leukemia cell-mediated model thatapparently does not exist in our IP administration model. Althoughprevious studies have been concerned with the counter-productive sideeffects resulting from elevated levels of IL-12-induced IFN-γ, severalcritical and beneficial roles for this cytokine have been demonstrated.Moreover in our model, a potentially problematic dampening of the immuneresponse was not observed, possibly due to the self-limiting nature ofthe leukemia cell-mediated therapy approach employed.

This work in a murine model of ALL using a LV constructed that engineersexpression of murine IL-12 has demonstrated that animals can becompletely protected from leukemia-induced death when certain levels ofIL-12 are produced by the transplanted cells

Example 2 Acute Myeloid Leukemia

The following myeloid leukemia lines were transduced with the murine LVIL-12 construct.

A lentiviral vector, (pHR-cPPT-EF1α-muIL-12-WPRE) that engineersexpression of murine interleukin-12 (mIL-12) was constructed andcharacterized. Plasmid pORF-mIL12 (IL-12elasti(p35::p40) Mouse(p35::p40)) was modified by creating EcoRI and BamHI restriction enzymessites, upstream and downstream of the muIL-12 gene, respectively, usinga QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla,Calif.). This resulting construct was then digested with EcoRI/BamHI(New England Biolabs). Murine IL-12 cDNA was purified afterelectrophoresis on a 1% agarose gel, and then subcloned into the pHR′ LVbackbone downstream of the elongation factor 1α promoter (EF1α).Positive plasmid clones for pHR-cPPT-EF1α-muIL-12-WPRE (i.e. LV-muIL-12)were identified by diagnostic restriction enzyme digestion analyses andsubsequent DNA sequencing (Innobiotech, Toronto, ON, Canada).

Lentivirus was produced by transfecting 293T cells with the plasmidspCMVΔR8.91, pMDG and either control enGFP lentivector(pHR-Cppt-'EF-GW-SIN). Viral supernatants were collected at 48 hourspost-transfection, filtered and concentrated by ultracentrifugation.Concentrated virus was serially diluted and the efficiency of viralproduction was assessed by detection of p24 antigen by ELISA.

To determine the transduction efficiency of mIL-12 lentivirus murinemyeloid leukemia lines MMB3.19 and C1498 (1 million cells/ml) wereinfected in vitro with mIL-12 or enGFP lentivirus at a multiplicity ofinfection (M.O.I) of 1. The infected cells were maintained at 37° C. andthe media changed 24 hours after culture. The supernatant was collected48 hours later and the levels of IL-12 measured by ELISA.

Cells transduced in vitro with lentivirus encoding mIL-12 produceefficiently high concentrations of IL-12 [˜214 ng/ml and 7.5 ng/ml forMMB3.19 and C1498 cells, respectively). MMB3.19-IL-12 and C1498-IL-12cells secreted 214 and 7.5 times more IL-12, respectively, than theenGFP transduced cells. The results are illustrated in the chart belowand in FIG. 8 a.

GFP MIL-12 MMB3.19 0.9 214.7 C1498 0.9 7.5

Example 3 Human IL-12 I. Lentiviral Vector Construction

Lentiviral vectors expressing human IL-12 cDNA were constructed by amethod similar to that described for mouse IL-12 construct. The cDNA ofhuman IL-12 was obtained as a fusion form from InvivoGen (pORF-hIL12(IL-12elasti(p35::p40)). The open reading frame of the gene wasamplified by the following PCR primers: hIL-12 ORF Fwd,5′-TTGGCGCGCCACCATGGGTCACCAGC-3′; and hIL-12 ORF Rev,5′-TTGGCGCGCCTTAGGAAGCATTCAGATAGCTCATCACTC-3′. The PCR product was thensubcloned into our Lentiviral backbone (pHR′-cPPT-EF1α-WPRE). Theconstruct was confirmed by diagnostic restriction enzyme digestionanalyses and subsequent DNA sequencing.

II. Transfection Experiment

To assess the pHR′-cPPT-EF1a-hIL12-WPRE construct, 1×10⁶ 293T cells weretransfected with the construct, the human IL-12 template pORF-hIL12 orempty lentivector pHR′-cPPT-EF1a-WPRE. Cell supernatant was collected 24and 48 hours after transfection. The hIL-12 level was measured by ELISA(BD pharmingen, San Diego, Calif.) (Chart below; FIG. 8 b).

Ill. Transduction to 293 T Cells

Lentivirus carrying hIL-12 open reading frame (LV-hIL-12) were producedby a transient triple-transfection method usingpHR-cPPT-EF1α-hIL-12-WPRE and accessory plasmids onto 293T monolayers bypolyethylenimine. Virus supernatant was collected 24 and 48 hours aftertransfection. To test the transduction ability of the LV-hIL1, 1×106293T cells were transduced with the virus supernatant. hIL-12 expressionlevel in the cell supernatant was measured by the same ELISA assay asmentioned above (Chart below FIG. 8 b).

IV. Transduction to AML.1 Cells

200-fold concentration of LV-hIL12 virus was obtained byultracentrifuge. To test the transduction ability of the virus to othertumor cell lines, 0.5 or 1 million of AML.1 cells (an acute leukemiacell line) were transduced with 1/100 diluted concentrated LV-hIL12virus. hIL-12 expression level in the cell supernatant was measured bythe same ELISA assay as mentioned above (FIG. 8 c).

24 h 48 h Ave (pg/ml) SD Ave (pg/ml) SD LV-hIL12 1010.052 33.145 840.39724.184 pHR-hIL12 774.131 340.254 933.513 50.522 pORF-hIL12 1079.43962.461 959.165 19.813 pHR vector 0 1.762 0 3.98LV-hIL12 will be used to transduce other human leukemic cell lines andprimary cancer cells derived from subjects with leukemia.

Example 4 Chronic Myeloid Leukemia in Humans

Immunotherapy offers a method to improve the treatment of leukemias, inparticular in combination with other treatment modalities. Indeed, maybeonly potent immune system-invoking therapy will be effective at fullyeradicating leukemia since residual disease often exists in patientsthat are in remission, which can be re-activated later. This isespecially true for chronic myeloid leukemia (CML), a clonal disorderinvolving the Philadelphia chromosome, which represents 15% of all adultleukemias. On the other hand, this delayed disease progression providesa key window of opportunity for immunotherapy. Since immunotherapy isnot dependent on abrogating cell functions by interrupting signaling oron intercalation into DNA by small molecules, for example, it can alsobe effective on transformed cells that are quiescent or inhabitinaccessible locales. Of importance, immunotherapy may be an effectiveway to target true cancer stem cells. Lastly, due to the circulating andsurveillance nature of the immune system, existing metastatic diseaseeven in primary CML patients could be treated by this approach.

Approximately 4500 new patients are diagnosed with CML in North Americaevery year. Onset of the most prevalent form of CML is associated with areciprocal translocation between chromosomes 9 and 22 leading to theformation of Bcr-Abl oncogene. This is manifested by a rapid expansionof bone marrow-derived hematopoietic cells of the myeloid lineage.Current first-line therapy involves treatment of CML patients withimatinib mesylate (Gleevec®), a small-molecule tyrosine kinase inhibitorof the Bcr-Abl product. Unfortunately, this is not a curative treatment.In fact, 4% of early-stage and a full 50% of advanced-stage CML patientsdevelop resistance to imatinib mainly due to ABL1 mutations (1).Imatinib another treatment, is also costly and requires life-longingestion of the drug; effects of prolonged administration (or of othersof this class) are not known. This strategy is also not likely to impactthe cancer stem cell, which may be relatively quiescent and therebyresistant to metabolic modulation. Also the lack of inhibitorspecificity for only the Bcr-Abl product means that other tyrosinekinases can also be affected. As such, imatinib has shown some seriousside effects; a recent study has shown that mice and human patientsreceiving imatinib demonstrate severe cardiotoxicity (2).

A wide range of immunotherapy strategies have been envisioned. Indeed,it has been known for years that the immune system is capable ofrecognizing and clearing cancer cells in some instances and yet not inothers. Cytokines have pleiotropic effects on the immune system. Onecytokine that has received a lot of attention towards amelioration ofcancer is interleukin-12 (IL-12). IL-12 is heterodimeric and acts toincrease antigen presentation by dendritic cells (DCs) and to inducetheir maturation. The basic concept behind therapy using IL-12 is thatit alerts the immune system to a higher degree of vigilance and if thisattention can be directed against cancer cells, elimination by theimmune system may be possible. IL-12 has been given as a systemic bolusfor treatment of leukemias but clinical outcomes have been quite modest.This may be due to difficulties in establishing appropriate dosing perpatient and the severe peripheral toxicities observed.

Interleukin-12 (IL-12).

IL-12 is a heterodimeric cytokine with multiple biological effects onthe immune system. It is composed of two subunits, p35 and p40, both ofwhich are required for the secretion of the p70 active form. IL-12 actson DCs, leading to increased maturation and antigen presentation, whichcan allow for the initiation of a T cell response against tumor specificantigens. It also drives the secretion of IL-12 by DCs, creating apositive feedback mechanism to amplify the response. Once a response isinitiated, IL-12 directs the immune system towards a Th1 cytokineprofile, inducing CD4⁺ T cells to secrete IFN-γ and leading to a CD8⁺cytotoxic T cell response (3). However, IL-12 is also a strongpro-inflammatory cytokine that leads to the secretion of other cytokinesincluding TNF-α which, combined with IFN-γ, is a prerequisite for thedevelopment of CD4+ cytotoxic T lymphocytes (CTL; ref. 4). Furthermore,IL-12 can promote the activation of macrophages and eosinophils throughinduction of IFN-γ and other cytokines. This leads to IL-12 secretionand further amplification of both the innate and acquired responses (3).However, high levels of IL-12, and consequently IFN-γ, have also beenassociated with induction of antagonistic molecules such as IL-10 andthe depletion of signalling molecules downstream of IL-12, such as STAT4(3, 5-7).

Lentiviruses (LVs) and Gene Therapy.

The first approved gene therapy clinical trial was published in 1989.Since then >2500 patients worldwide have received gene therapy to date.

Safety is a high priority. One major vector system that has beenresponsible for generating renewed enthusiasm is based on LVs. LV aremost-commonly derived from HIV-1 (17). Substantial segments of the viralgenome have been deleted and additional safety elements, such asself-inactivating LTRs, have been added (18). Moreover, these vectorsare now produced in ways to reduce the possibility of recombinationdeveloping replication competent lentivirus (RCL). Indeed, substantialeffort has gone into testing the safety and efficacy of this platform;for example, LVs offer stable integration but with less insertion intopromoters that can disrupt cell functions than occurs withonco-retroviruses. LVs also allow the ability to engineer coexpressionof more than one gene. A number of these bicistronic constructs havebeen generated by the inventors (see ref. 19, 20). The LV constructscomprise a novel suicide control system. This enzyme/prodrug combinationemploys a modified human enzyme engineered to respond to AZT (ref. 21;see Comment (ref. 22). This safety system offers the ability to controlthe fate of transduced cells and will be practical to use in any settinginvolving transplant of tumor cells, stem cells, and the like.

Safety improvements and the efficiency of LVs have recently led toclinical trials. The first LV trial was been completed in 2006 andinvolved anti-sense RNA sequences as transgenes that targeted HIV (23).This study was performed in AIDS patients with high viral loads; somereductions in these viral loads were observed. More importantly, no RCLwas found between the recombinant vector and the endogenous wild-typevirus. These results have now led to at least 6 other LV protocols beinginitiated for indications including cancer and inherited disease. Suchoutcomes have also led to a renaissance in corporate interest in genetherapy that still has a large but untapped potential to treat a varietyof disorders.

As the inventors have found, localized concentration of IL-12 at thetumor/DC/T cell interface may be relevant for up-regulation of theimmune response, and effective dosing at that site is not beinggenerated in the clinical protocols.

State-of-the-art gene transfer techniques (lentiviruses; LVs) were usedto quantitatively modulate the expressed IL-12 profile by the tumor cellitself. LVs are very efficient at stably transferring genes into cells.

The inventors have generated a novel clinically-oriented LV thatengineers expression of human IL-12. Virus has been produced and virusand the vector have been validated in established human cancer celllines by quantitating titer and human IL-12 production (see FIG. 8 b).Human primary CML cells will be transduced which will produce varyinglevels of human IL-12. The cells will be analysed to demonstrate thatthe human IL-12 produced by the tranduced cells is functional. Apre-clinical xenograft model will be adapted to examine maintenance ofthe transduced CML cells. The kinetics of human IL-12 produced in vivowill be measured.

Gleevec is the treatment of choice; however side effects, resistance,the need for long-term therapy, and high cost are associated withGleevac use.

Murine Models of CML.

Two established CML lines were tested and show differential productionof IL-12 in vitro in transduced populations derived from these lines.

CML and ALL are similar in that high remission rates in adults arefollowed by high relapse rates. This clinical course not only providesinitial material suitable for infecting with the vector constructsdescribed herein but a rationale for subsequent treatment. Importantly,CML shows this bi/tri-phasic progression and some initial response toimatinib that allows time to develop immune modulating tumor cellsfollowing vector transductions.

LVs offer some real advantages over other gene transfer methods thatseek to generate stable cell lines secreting IL-12 for suchapplications: for example—plasmid transfection is very inefficient andadenovirus- or AAV-mediated gene delivery do not lead to appreciablevector integration, which will provide variable levels of IL-12 overtime. The inventors have shown that transduced murine cells stablyexpress transgenes ˜2 years after initial infection (24).

Synthesis of Human Vector.

A recombinant LV that engineers stable expression of human IL-12 wasgenerated. The cDNA for human IL-12 was obtained as a fusion form fromInVivoGen (pORF with IL-12elasti(p40::p35)). This cDNA was subcloned asabove into the pHR′ LV backbone. Diagnostic restriction digests andsequencing of both DNA strands was performed to confirm the fidelity ofthe new construct. This first construct will be monocistronic; otherconstructs may employ our suicide strategy involving mutated thymidylatekinase mentioned above (21) that would add another layer of safety.

Generation of High-Titer Vector Stocks.

High titer recombinant virion stocks were generated and titered invitro. High titer vector stocks were established by ultracentrifugationof collected and pooled supernatants after triple plasmid transfectionsof 293T cells as done before (20). The vector was pseudotyped with theVSV-g glycoprotein which allows a wide range of cells to be infected.After sufficient titer of the pHR′ human IL-12 delivery vector isobtained, pooled vector stocks will be tested by a ‘Direct’ assay toensure that RCL has not been generated. In this assay, recipient 293Tcells are infected a single time and then grown out for a number ofpassages. After 4-6 weeks, supernatants from these infected cells arecollected and used to infect naïve cells. These cells are grown out andthen assayed by functional assays and PCR on isolated genomic DNA todetermine if vector has been functionally transmitted to these secondaryrecipient targets.

Testing in 293T Cells.

The level of human IL-12 produced in comparison to vector copy number ininfected cells will be determined. Firstly, 293T cells will be infectedat a range of modest MOIs from about 0.1 to 100. Supernatants from poolsof infected cells, done in triplicate, will be examined for human IL-12production by ELISAs. Next, individual cell clones will be establishedby limiting dilution. These cell lines will examined for human IL-12production relative to copies of integrated provirus—as measured bySouthern blots. Controls will be comprised of 293T cells infected with aLV/eGFP viruspreviously constructed (19). This information will provideinformation relating to the relative MOIs to be used and allowscorrelation of the secretion of this human form of IL-12 with relativevector copy number. Use of this stable cell line will provide areference point for titering all future viral preparations that are madewith the intent of infecting patient CML cells, which may haveconsiderable variability in sample-to-sample infection frequencies.

Testing in Human CML.

Firstly, established CML cell lines will be infected at various MOIs andclonal populations will be assessed for IL-12 expression in relation tovector copy number. It has been shown by the inventors that K562 (a CMLline) is readily and productively infected with recombinant LVs (21).Numerous clones from each pool will be derived and examined for vectorcopy and relative human IL-12 production. Cell viability of clonesproducing various levels of human IL-12 over time will be measured bythymidine incorporation assays. Cells will be cultured for many weeksand compared with original clones frozen initially after limitingdilution to determine if human IL-12 production changes over time.Vector stability will also be measured in these cells by repeat SouthernBlot analyses. Secondly, primary human CML cells will be obtained from aminimum of 3-5 CML donors initially to reduce reliance on a singlesample. Here cells will be infected at 2 or 3 different MOIs. Cells fromeach donor will be handled separately to give information on thevariability that can be expected. As above, human IL-12 production willbe measured by ELISA in relation to vector copy number.

Additional pre-clinical data will be obtained. From a number oftransduced K562 and Jurkat clonal lines, the sequence of the human IL-12cDNA from the integrated provirus in genomic DNA will be determinedafter PCR amplification and subcloning to a stable plasmid. This willprovide information on the stability of the vector itself and whetherrecombinations are occurring that could decrease protein expressionlevels from a given vector copy number. If consistent alterations areobserved in a variety of clones such sequences could be mutated toreduce overlap or alter secondary mRNA structure to favor maintenance offidelity. Further the vector integration site of cell populations byLM-PCR will be analysed to determine clonality. It will also beimportant to determine that the human IL-12 secreted by the transducedCML clones is functional. For this primary human DC cultures will beused to examine stimulation and the enhancement of T cell proliferationcompared to controls.

It will be determined whether vector-transduced primary CML cells thathave undergone growth arrest (by very high dose irradiation, forexample) in preparation for safe clinical infusions into patients arestill able to secrete similar levels of human IL-12 compared to controlcells. No differences are expected as others have shown stableexpression of GM-CSF and CD40L, for example, in patient leukemia cellsafter irradiation (25). One group even reported enhanced transgeneexpression in leukemia cells after γ-irradiation (26). Also, the suicidegene component mentioned above may be added, and killing efficiency ofbicistronically transduced primary CML cells producing human IL-12 willbe assessed after AZT addition at concentrations we have used before(21).

Test CML Cell Growth In Vivo.

The cell lines are assessed for growth in vivo. Cells will be introducedin immune deficient NOD/SCID mice and mice will be examined for thepersistence of transduced CML cell lines and primary patient cells invivo in this xenograft model. This model shows stable engraftment ofhuman hematopoietic cells, especially when an antibody is given toreduce murine NK cell activity. anti-CD122 antibody (24) from ahybridoma cell line is purified in milligram quantities (FIG. 9). Bothgrowth-arrested cells and un-manipulated transduced cells will be givenat various doses to recipient NOD/SCID mice. Persistence of transducedCML cells will be determined by conventional assays involving flowcytometry for human cell surface antigens (such as CD45/CD71) along withRT-PCR analyses for the LV as has been done for the Bcr-Abl oncogenefusion (27). These studies will be important to prove that the CML cellscomprise the primary populations in the xenografted animals. As well,circulating levels of human-specific IL-12 will be determined by ELISA;production of secondary cytokines such as IFN-γ is also measured.

Where the bicistronic vector that engineers expression of the novelsuicide gene is employed, the effectiveness of transduced cell killingin vivo can be measured after the addition of AZT to animals—dosing thatis below the level of systemic toxicity is described in (21). A fullyadaptive transplant system in this xenograft model is developed whereinmatching genetically modified cells are returned to animals previouslyreconstituted with autologous patient hematopoietic components. Theoptimal dose of IL-12 relative to immune response is determined. Theeffect of the addition of other co-stimulatory molecules or alternativecytokines that perturb the immune response invoked either positively ornegatively are assessed. Lentivectors that express shRNAs thatdownregulate expression of important genes that may effect stimulationsuch as IL-10 are also assessed. The contribution of various populationsof hematopoietic cells themselves using depletion and sorting-mediatedadd-back studies are also assessed.

Example 5

Leukemia cells from 4 donors from each group (CML, AML, CLL, ALL) willbe enriched following Ficoll centrifugation by established protocols.Initially, for AML and ALL we will carefully select patients with highleukocyte (>60k) and high % blast counts in which case we expectenrichments to exceed 95% purity. For CML, patients in blast crisis willbe selected to achieve the same result. For CLL mature CLL lymphocytesfrom patients with very high leukocyte counts (>100k) will be achievedto achieve this enrichment. In each experiment, the leukemia cellpopulation will be infected at 3 different MOIs using our LV/huIL-12construct and a LV/enGFP control. An enzyme-linked immunospot (ELISPOT)assay for use as a readout in these experiments is being developed. Thecloned, stable, murine lines produce a range of IL-12 from 200-40000pg/10⁶/ml/2 hrs and serve to calibrate the ELISPOT assay by correlatingspot size to known secretion levels at the signal cell level. A similarcalibration set will be created with human established cell lines bysubcloning after the primary LV/huIL12 transduction. The ELISPOT assaywill allow us to quantify not only the percentage of primary leukemiacells expressing IL-12 from the transduced IL-12 vector, but also willprovide a distribution of IL-12 production levels. The assay will bedeveloped to reliably yield at least 10% of the leukemia cellsexpressing at least 20000 pg/10⁶/ml/2 hr. Primary cells will be frozenand thawed and retested to determine the stability of this distribution.Primary cells will also be irradiated and retested for the productionand distribution of IL-12 levels. Clinical protocols using thesepopulations would serve as autologous cell based vaccines to be used toprevent relapse in patients who achieve CR.

Example 6 Acute Lymphoblastic Leukemia (ALL)

Similarly as described for CML, ALL cells transduced with a LV IL-12construct will be made and tested.

Testing in Human ALL Cells.

Firstly, established ALL cell lines will be infected at various MOIs andclonal populations will be assessed for IL-12 expression in relation tovector copy number. It has been shown by the inventors that Jurkat cells(an ALL line) are readily and productively infected with recombinant LVs(21). Numerous clones from each pool will be derived and examined forvector copy and relative human IL-12 production. Cell viability ofclones producing various levels of human IL-12 over time will bemeasured by thymidine incorporation assays. Cells will be cultured formany weeks and compared with original clones frozen initially afterlimiting dilution to determine if human IL-12 production changes overtime. Vector stability will also be measured in these cells by repeatSouthern Blot analyses. Secondly, primary human ALL cells are obtainedfrom a minimum of 3-5 ALL donors initially to reduce reliance on asingle sample. Here cells are infected at 2 or 3 different MOIs. Cellsfrom each donor are handled separately to give information on thevariability that can be expected. As above, human IL-12 production willbe measured by ELISA in relation to vector copy number.

Additional pre-clinical data will be obtained. From a number oftransduced K562 and Jurkat clonal lines, the sequence of the human IL-12cDNA from the integrated provirus in genomic DNA will be determinedafter PCR amplification and subcloning to a stable plasmid. This willprovide information on the stability of the vector itself and whetherrecombinations are occurring that could decrease protein expressionlevels from a given vector copy number. If consistent alterations areobserved in a variety of clones such sequences could be mutated toreduce overlap or alter secondary mRNA structure to favor maintenance offidelity. Further the vector integration site of cell populations byLM-PCR will be analysed to determine clonality. It will also beimportant to determine that the human IL-12 secreted by the transducedCML clones is functional. For this primary human DC cultures will beused to examine stimulation and the enhancement of T cell proliferationcompared to controls.

It will be determined whether vector-transduced primary ALL cells thathave undergone growth arrest (by very high dose irradiation, forexample) in preparation for safe clinical infusions into patients arestill able to secrete similar levels of human IL-12 compared to controlcells. No differences are expected as others have shown stableexpression of GM-CSF and CD40L, for example, in patient leukemia cellsafter irradiation (25). One group even reported enhanced transgeneexpression in leukemia cells after γ-irradiation (26). Also, the suicidegene component mentioned above is optionally added, and killingefficiency of bicistronically transduced primary ALL cells producinghuman IL-12 will be assessed after AZT addition at concentrations wehave used before (21).

Administering IL-12 Expressing Cells to an ALL Subject

Acute Lymphoblastic Leukemia:

It is estimated that 5,200 new patients will be diagnosed with ALL inthe US in 2007, and 1,420 will die of the illness. ALL is the most isthe most common type of leukemia in children with 61% of diagnoses madein individuals under age 20 (29). The overall 5-year relative survivalrate for the period 1996-2003 was 64.0%. There was a slightly positiveannual percentage change (0.3%) in ALL incidence for the period of1985-2005 (29).

The malignant hematopoietic cells are lymphoid precursor cells.Cytogenetic abnormalities occur in ˜70% of cases of ALL in adults butare not associated with a single translocation event as in CML. Thestandard treatment course has been given the terms induction,consolidation, maintenance, and CNS prophylaxis—but even with intensivetherapy only 20-40% of adults with ALL are cured with current regimens.Therapy for ALL includes conventional chemotherapy (vincristine,anthracycline, cyclophosphamide, L-asparaginase etc.), radiation therapyand bone marrow transplant. Newer drugs have been developed includingclofarabine, nelarabine, and dasatinib, but here responses have beenrelatively modest and toxicities remain an issue.

Imatinib has also been used in Philadelphia chromosome positive ALL.Imatinib has limited effectiveness in ALL treatment when used as asingle agent, but several studies have shown improved outcomes when itis combined with standard chemotherapy (30). Clofarabine (Clolar®) wasapproved in December of 2004 for pediatric patients with relapsed orrefractory ALL overall response rates average 25% (30). Nelarabine(Arranon®) was approved as an orphan drug by the FDA in October, 2005for treatment of T-cell ALL. Complete responses are reported in 54% ofpatients with T-cell ALL (30). Approximately 700 ALL patients per yearin the US have T-cell ALL (30).

Drugs in development for ALL include Rituximab in Phase III, AMN107 and852A both in Phase II, Nilotinib (Tasigna®) and AT9283 both in PhaseI/II and KW-2449 in Phase I. Cell based therapies such asnonmyeloablative stem cell transplant and allogeneic umbilical cordblood transplantation are also in development. Drugs in trials forspecific types of ALL include therapeutics directed to T-cell ALL(T-ALL) such as Alemtuzumab (Campath®), daclizumab and denileukindiftitox (Ontak®) all in Phase II and Similarly, a number of CML drugsin trials for Ph+ ALL such as MK0457 and Bortezomib (Velcade®) which areboth in Phase II, SKI-606 in Phase I/II and INNO-406 in Phase I.

Clinical Use

50 ml of heparanized blood is collected from patients following REBapproved informed consent. The blood is diluted with 110 ml of alphamedium and aliquoted in to 50 ml conical centrifuge tubes. Ficol hypaqueis injected under the blood and the tubes are spun at 1600 rpm at 15 Cfor 20 minutes. The layer of mononuclear cells is removed andresuspended in 100 ml alpha medium with 5% FCS. The cells are spun at1000 rpm for 10 minutes and then resuspended in 10 ml alpha medium with5% FCS cells are then counted and then frozen for future use ordistributed for fresh experiments. This would yield over 1×109 blastsfrom the peripheral blood of patients.

Blast cells are collected from the subject prior to chemotherapy whenthey are very high in numbers. The cells or a portion thereof areoptionally frozen. The patient is treated with chemotherapy or otherappropriate modality. Cells are then thawed if frozen, infected with LVIL-12 and analyzed for the required level of expression (e.g thethreshold level). Cells meeting this criteria are optionally irradiated,and reintroduced into the patient.

Where the vector construct comprises a safety gene component, cells areoptionally not irradiated.

Further cells are optionally infected prior to freezing.

Administering IL-12 Expressing Cells to a Subject with CML

Chronic Myeloid Leukemia:

It is estimated that 4,570 people in the US will be diagnosed with CMLand 490 will die of this illness 2007 (30). There was a negative annualchange in incidence (−2.6%) of CML for the period of 1997-2004 (30).

Current preferred first-line therapy involves treatment of CML patientswith imatinib mesylate (Gleevec®). It has been reported that 4% ofearly-stage CML patients and a full 50% of advanced-stage CML patientsdevelop resistance to imatinib (32). Imatinib mesylate treatment alsorequires life-long medication; the full effects of such prolongedadministration of this agent (or others of this class) are not yetknown. Gleevec can cause severe side effects such as cytopenias,particularly anemia, neutropenia, and thrombocytopenia; severecongestive heart failure and left ventricular dysfunction; severehepatotoxicity; grade 3/4 hemorrhage and gastrointestinal perforationsincluding some that have been fatal. Along those lines, a recent studyhas shown that mice and human patients receiving imatinib mesylatedemonstrate cardiotoxicity (2); although the overall prevalence of thisseverely adverse event has not yet been systematically verified andaccurately quantitated.

Dasatinib (Sprycel®) has recently been introduced as a therapy for CMLpatients that have failed treatment with imatinib. Dasatinib can alsoproduce severe and sometimes fatal side effects: thrombocytopenia,neutropenia, and anemia (NCI CTC Grade 3 or 4); severe hemorrhagesincluding fatalities have occurred in a significant percentage ofpatients (1-7% depending on site of hemorrhage). Most bleeding eventswere associated with severe thrombocytopenia. Other side effects includesevere fluid retention and cardiac effects (QT prolongation) (33).

Nilotinib (Tasigna®) has very recently been approved in the US as a newanti-cancer therapy for CML patients who are resistant or intolerant totreatment with imatinib. Similar to dasatinib, nilotinib can causeneutropenia and thrombocytopenia. Nilotinib also prolongs the QTinterval and sudden deaths have been reported (34).

Other treatment options for patients with CML include conventionalcytotoxic chemotherapy, interferon-alpha, bone marrow transplant andallogeneic stem cell transplant.

Drugs in development for CML include Lonafarnib Phase III, LBH589 PhaseII/111, AT9283 Phase I/II, MK0457 Phase II, Bortezomib (Velcade) PhaseII. 852A Phase II, SKI-606 Phase I/II, allogeneic umbilical cord bloodtransplantation Phase II, XL228 Phase I, KW-2449 Phase I, INNO-406 PhaseI and homoharringtonine (Ceflatonin®) which has recently completed PhaseI/II (35).

Clinical Use

50 ml of heparanized blood is collected from patients following REBapproved informed consent. The blood is diluted with 110 ml of alphamedium and aliquoted in to 50 ml conical centrifuge tubes. Ficol hypaqueis injected under the blood and the tubes are spun at 1600 rpm at 15 Cfor 20 minutes. The layer of mononuclear cells is removed andresuspended in 100 ml alpha medium with 5% FCS. The cells are spun at1000 rpm for 10 minutes and then resuspended in 10 ml alpha medium with5% FCS cells are then counted and then frozen for future use ordistributed for fresh experiments. This would yield over 1×109 blastsfrom the peripheral blood of patients.

Blast cells are collected from the subject prior to chemotherapy whenthey are very high in numbers. The cells or a portion thereof areoptionally frozen. The patient is treated with chemotherapy or otherappropriate modality. Cells are then thawed if frozen, infected with LVIL-12 and analyzed for the required level of expression (e.g thethreshold level). Cells meeting this criteria are optionally irradiated,and reintroduced into the patient.

Where the vector construct comprises a safety gene component, cells areoptionally not irradiated.

Further cells are optionally infected prior to freezing.

Administering LV IL-12 to a CLL Patient

CLL

B-CLL is the most common leukemia of adults with an expectation of˜16500 cases in NA this year (Estimates based on American Cancer Societyand Canadian Cancer Society Reports). Remissions can be achieved withpurine analogues and monoclonal antibody therapy however the diseasesinvariable progresses. Allogeneic stem cell transplants can be curativebut many patients do not qualify for this treatment because of theirage. The observation that GVL responses occur after stem celltransplantation confirms that an anti-leukemia immune response to CLL ispossible. The slow progression of B-CLL also makes this diseaseattractive for immunotherapy approaches.

Clinical Use

50 ml of heparanized blood is collected from patients following REBapproved informed consent. The blood is diluted with 110 ml of alphamedium and aliquoted in to 50 ml conical centrifuge tubes. Ficol hypaqueis injected under the blood and the tubes are spun at 1600 rpm at 15 Cfor 20 minutes. The layer of mononuclear cells is removed andresuspended in 100 ml alpha medium with 5% FCS. The cells are spun at1000 rpm for 10 minutes and then resuspended in 10 ml alpha medium with5% FCS cells are then counted and then frozen for future use ordistributed for fresh experiments.

This would yield over 1×109 blasts from the peripheral blood ofpatients.

Blast cells are collected from the subject prior to chemotherapy whenthey are very high in numbers. The cells or a portion thereof areoptionally frozen. The patient is treated with chemotherapy or otherappropriate modality. Cells are then thawed if frozen, infected with LVIL-12 and analyzed for the required level of expression (e.g thethreshold level). Cells meeting this criteria are optionally irradiated,and reintroduced into the patient.

Where the vector construct comprises a safety gene component, cells areoptionally not irradiated.

Further cells are optionally infected prior to freezing.

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Differential    effect of high doses versus low doses of interleukin-12 on the    adoptive transfer of human specific cytotoxic T lymphocyte in    autologous lung tumors engrafted into severe combined    immunodeficiency disease-nonobese diabetic mice: relation with    interleukin-10 induction. Cancer. 91:113-122 (2001).-   15. Gollob J A, Mier J W, Veenstra K, McDermott D F, Clancy D,    Clancy M and Atkins M B. Phase I trial of twice-weekly intravenous    interleukin 12 in patients with metastatic renal cell cancer or    malignant melanoma: ability to maintain IFN-gamma induction is    associated with clinical response. Clin. Cancer Res. 6:1678-1692    (2000).-   16. Hoshino T, Jiang Y Z, Dunn D, Paul D, Qazilbash M, Cowan K, Wang    J, Barrett J and Liu J. Transfection of interleukin-12 cDNAs into    tumor cells induces cytotoxic immune responses against native tumor:    implications for tumor vaccination. Cancer Gene Ther. 5:150-157    (1998).-   17. Vigna E and Naldini L. 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Engineered human tmpk/AZT as a novel enzyme/prodrug axis for    suicide gene therapy. Mol. Ther. 15:962-970 (2007).-   22. Baum C. I could die for you: new prospects for suicide in gene    therapy. Mol. Ther. 15:848-849 (2007).-   23. Levine B L, Humeau L M, Boyer J, MacGregor R R, Rebello T, Lu X,    Binder G K, Slepushkin V, Lemiale F, Mascola J R, Bushman F D,    Dropulic B and June C H. Gene transfer in humans using a    conditionally replicating lentiviral vector. Proc. Natl. Acad. Sci.    U.S.A. 103:17372-17377 (2006).-   24. Yoshimitsu M, Higuchi K, Ramsubir S, Nonaka T, Rasaiah V I,    Siatskas C, Liang S B, Murray G J, Brady R O and Medin J A.    Efficient correction of Fabry mice and patient cells mediated by    lentiviral transduction of hematopoietic stem/progenitor cells. Gene    Ther. 14:256-265 (2007).-   25. Dessureault S, Noyes D, Lee D, Dunn M, Janssen W, Cantor A,    Sotomayor E, Messina J and Antonia S J. A phase-I trial using a    universal G M-CSF-producing and CD40L-expressing bystander cell line    (GM.CD40L) in the formulation of autologous tumor cell-based    vaccines for cancer patients with stage IV disease. Ann. Surq.    Oncol. 14:869-884 (2007).-   26. Vereecque R, Saudemont A, Wickham T J, Gonzalez R, Hetuin D,    Fenaux P and Quesnel B. Gamma-irradiation enhances transgene    expression in leukemic cells. Gene Ther. 10:227-233 (2003).-   27. Eisterer W, Jiang X, Christ O, Glimm H, Lee K H, Pang E, Lambie    K, Shaw G, Holyoake T L, Petzer A L, Auewarakul C, Barnett M J,    Eaves C J and Eaves A C. Different subsets of primary chronic    myeloid leukemia stem cells engraft immunodeficient mice and produce    a model of the human disease. Leukemia. 19:435-441 (2005).-   28. Del Vecchio M, Bajetta E, Canova S, Lotze M T, Wesa A, Parmiani    G and Anichini A. Interleukin-12: biological properties and clinical    application. Clin. Cancer Res. 13:4677-4685 (2007).-   29. National Cancer Institute. SEER Cancer Statistics Review. 2007:    (2007).-   30. Seiter K. Acute Lymphoblastic Leukemia. (2006).-   31. Redaelli A, Stephens J M, Laskin B L, Pashos C L and Botteman    M F. The burden and outcomes associated with four leukemias: AML,    ALL, CLL and CML. Expert Rev. Anticancer Ther. 3:311-329 (2003).-   32. Piccaluga P P, Martinelli G and Baccarani M. Advances in the    treatment for haematological malignancies. Expert Opin.    Pharmacother. 7:721-732 (2006).-   33. Dasatinib (Sprycel®) Drug Product Label.-   34. Nilotinib (Tasigna®) Drug Product Label.-   35. ChemGenix Pharmaceuticals. ChemGenix Pharmaceuticals Press    Release (2007).-   36. Centers for Medicare & Medicaid Services, the US Department of    Health and Human Services. 2004 Report. (2004).-   37. Frost & Sullivan. U.S. Gene Therapy Markets. (2005).-   38. Gene Therapy Clinical Trials Worldwide online database    maintained by the Journal of Gene Medicine. Gene therapy clinical    trials numbers query search. (2007).

Example 7 Treating Solid Tumors

Solid tumors are removed partially of fully from a subject. The solidtumor is optionally any respectable tumor. The tumor is optionally arenal cell cancer, melanoma or prostate cancer.

Single cell suspensions are obtained and cells are transduced ortransfected with an IL-12 vector contrusct such as LV hIL-12.Transfected or transduced cells are optionally irradiated to inducegrowth arrest and prevent cell division. Transduced cells comprisingvector constructs comprising an activator polynucleotide such as amodified tmpk molecule are not irradiated as cells expressing theactivator polynucleotide can be killed by administration of the prodrug.

A population of cells including transduced cancer cells is administeredto the subject from which the cancer was derived. The population ofcells is administered, intradermally or subcutaneously about once aweek, once every two weeks, or about once a month for a 3 month period.Approximately 1×10⁶ to 1×10⁸ cells are administered.

The subject is monitored for an anti-cancer immune response and cancerprogression.

Example 8 Research Models and Systems

Determine the Critical Aspects of Initiating Anti-Leukemia Responses inthe Murine System.

The in vivo induction of anti-leukemia immunity using in vitro modelswill be studied. DCs mature in culture when exposed to 70Z/3-IL-12 cellsonly in the presence of spleen cells. Untransduced 70Z/3 cells do notmirror this effect. Selected populations of spleen cells will besystematically removed to determine which spleen cells are responsiblefor the observed effects. Antibodies specific for subpopulations of Tcells, NK cells, and macrophages, will be used in combination witheither MACS or FACS for depletion and/or enrichment. These experimentswill be conducted in transwell plates which allow the physicalseparation of the various cell types to identify critical cell-cellinteractions. DC maturation (increased expression of CD80) as our primeread out has been used. However, it is possible that DC maturation inthe presence of 70Z/3 cells will be followed by activation of specific Tcell populations. The in vitro system will be used to determine if Tcell responses are initiated and, if so, the nature of those responses.Cytokine production typical of Th1 induction (such as IFNγ) as well asthe appearance CD4⁺ and or CD8⁺ mature T cells specific for 70Z/3 cellswill be monitored. 70Z/3 specific T cell clones will be expanded andtheir cell surface phenotype will be characterized. Their cytotoxicpotential in Cr⁵¹ release assays using 70Z/3 cells as targets will betested.

The established in vivo model will also be used to explore the inductionof protective immunity. In particular, adoptive transfer experimentswill be undertaken to determine if CD4⁺ cells can confer immunity and ifso if these cells are CD4⁺ CTL or NKT cells. These cells will beisolated and cloned in vitro after they arise in the mice to establishtheir growth properties and mechanism of cytotoxicity. By comparing theinduction of immunity to AML to our current ALL model, we will study whysome cancers are more immunogenic that others.

With this background knowledge we will initiate IL-12 transductionexperiments using established human leukemia cell lines representingdifferent classes of leukemia. These include K562, CES1, OCIAML1,OCIAML2, Jurkat, Raji. The Medin lab has already shown that both K562and Jurkat are readily infected with LV vectors in past experiments. Thecell lines will be transduced in bulk culture after which clones will beselected by limit dilution. The clones will be examined for cellproliferation by thymidine incorporation assays and for IL-12 productionby ELISA. The stability of the IL-12 production will be determined afterextended cell culture times as well as after several freeze/thaw cycles.Repeat Southern blot analysis will be used to determine vector copynumber and stability as well.

Human In Vitro Assay.

Established cell lines and primary samples will also be used to developin vitro assays similar to those underway in the murine system. In vitroculture conditions that support human DCs and T cell subsets have beendeveloped. Using these as a starting point the effects of IL-12producing cell lines and primary samples in short term assays will bemonitored. We will establish the ability of IL-12 producing cell linesand primary leukemia samples to influence the maturation of human DCs inthe presence and absence of selected T cell subsets. We will monitorcell surface markers such as CD80 for DC maturation and IFNγγ secretionfor induction of Th1 responses. If evidence of an IR is detected, CD4and CD8 subsets will be isolated and tested for anti-leukemiacytotoxicity and specificity.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

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Sequences pHR′.cPPT.EF.CD19ΔTmpkF105YR200A.WPRE.SIN. SEQ ID NO: 1AATTACCTGTGGTTTCATTTACTCTAAACCTGTGATTCCTCTGAATTATTTTCATTTTAAAGAAATTGTATTTGTTAAATATGTACTACAAACTTAGTAGTTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAGAACTACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGGTAGAAGAGGCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGAGAGAAGTGTTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTGATATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTATCGATAAGCTTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCATGCCACCTCCTCGCCTCCTCTTCTTCCTCCTCTTCCTCACCCCCATGGAAGTCAGGCCCGAGGAACCTCTAGTGGTGAAGGTGGAAGAGGGAGATAACGCTGTGCTGCAGTGCCTCAAGGGGACCTCAGATGGCCCCACTCAGCAGCTGACCTGGTCTCGGGAGTCCCCGCTTAAACCCTTCTTAAAACTCAGCCTGGGGCTGCCAGGCCTGGGAATCCACATGAGGCCCCTGGCATCCTGGCTTTTCATCTTCAACGTCTCTCAACAGATGGGGGGCTTCTACCTGTGCCAGCCGGGGCCCCCCTCTGAGAAGGCCTGGCAGCCTGGCTGGACAGTCAATGTGGAGGGCAGCGGGGAGCTGTTCCGGTGGAATGTTTCGGACCTAGGTGGCCTGGGCTGTGGCCTGAAGAACAGGTCCTCAGAGGGCCCCAGCTCCCCTTCCGGGAAGCTCATGAGCCCCAAGCTGTATGTGTGGGCCAAAGACCGCCCTGAGATCTGGGAGGGAGAGCCTCCGTGTGTCCCACCGAGGGACAGCCTGAACCAGAGCCTCAGCCAGGACCTCACCATGGCCCCTGGCTCCACACTCTGGCTGTCCTGTGGGGTACCCCCTGACTCTGTGTCCAGGGGCCCCCTCTCCTGGACCCATGTGCACCCCAAGGGGCCTAAGTCATTGCTGAGCCTAGAGCTGAAGGACGATCGCCCGGCCAGAGATATGTGGGTAATGGAGACGGGTCTGTTGTTGCCCCGGGCCACAGCTCAAGACGCTGGAAAGTATTATTGTCACCGTGGCAACCTGACCATGTCATTCCACCTGGAGATCACTGCTCGGCCAGTACTATGGCACTGGCTGCTGAGGACTGGTGGCTGGAAGGTCTCAGCTGTGACTTTGGCTTATCTGATCTTCTGCCTGTGTTCCCTTGTGGGCATTCTTCATCTTGCCGGCGGGGCTGCAGGGATGGCGGCCCGGCGCGGGGCTCTCATAGTGCTGGAGGGCGTGGACCGCGCCGGGAAGAGCACGCAGAGCCGCAAGCTGGTGGAAGCGCTGTGCGCCGCGGGCCACCGCGCCGAACTGCTCCGGTTCCCGGAAAGATCAACTGAAATCGGCAAACTTCTGAGTTCCTACTTGCAAAAGAAAAGTGACGTGGAGGATCACTCGGTGCACCTGCTTTTTTCTGCAAATCGCTGGGAACAAGTGCCGTTAATTAAGGAAAAGTTGAGCCAGGGCGtGACCCTCGTCGTGGACAGATACGCATTTTCTGGTGTGGCCTACACaGGTGCCAAGGAGAATTTTTCCCTAGACTGGTGTAAACAGCCAGACGTGGGCCTTCCCAAACCCGACCTGGTCCTGTTCCTCCAGTTACAGCTGGCGGATGCTGCCAAGCGGGGAGCGTTTGGCCATGAGCGCTATGAGAACGGGGCTTTCCAGGAGCGGGCGCTCCGGTGTTTCCACCAGCTCATGAAAGACACGACTTTGAACTGGAAGATGGTGGATGCTTCCAAAAGCATCGAAGCTGTCCATGAGGACATCCGCGTGCTCTCTGAGGACGCCATCGCCACTGCCACAGAGAAGCCGCTGGGGGAGCTATGGAAGTGAGGATCCAAGCTTCAATTGTGGTCACTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGCTCGAGACCTAGAAAAACATGGAGCAATCACAAGTAGCAATACAGCAGCTACCAATGCTGATTGTGCCTGGCTAGAAGCACAAGAGGAGGAGGAGGTGGGTTTTCCAGTCACACCTCAGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGCCTTGACATTATAATAGATTTAGCAGGAATTGAACTAGGAGTGGAGCACACAGGCAAAGCTGCAGAAGTACTTGGAAGAAGCCACCAGAGATACTCACGATTCTGCACATACCTGGCTAATCCCAGATCCTAAGGATTACATTAAGTTTACTAACATTTATATAATGATTTATAGTTTAAAGTATAAACTTATCTAATTTACTATTCTGACAGATATTAATTAATCCTCAAATATCATAAGAGATGATTACTATTATCCCCATTTAACACAAGAGGAAACTGAGAGGGAAAGATGTTGAAGTAATTTTCCCACAATTACAGCATCCGTTAGTTACGACTCTATGATCTTCTGACACAAATTCCATTTACTCCTCACCCTATGACTCAGTCGAATATATCAAAGTTATGGACATTATGCTAAGTAACAAATTACCCTTTTATATAGTAAATACTGAGTAGATTGAGAGAAGAAATTGTTTGCAAACCTGAATAGCTTCAAGAAGAAGAGAAGTGAGGATAAGAATAACAGTTGTCATTTAACAAGTTTTAACAAGTAACTTGGTTAGAAAGGGATTCAAATGCATAAAGCAAGGGATAAATTTTTCTGGCAACAAGACTATACAATATAACCTTAAATATGACTTCAAATAATTGTTGGAACTTGATAAAACTAATTAAATATTATTGAAGATTATCAATATTATAAATGTAATTTACTTTTAAAAAGGGAACATAGAAATGTGTATCATTAGAGTAGAAAACAATCCTTATTATCACAATTTGTCAAAACAAGTTTGTTATTAACACAAGTAGAATACTGCATTCAATTAAGTTGACTGCAGATTTTGTGTTTTGTTAAAATTAGAAAGAGATAACAACAATTTGAATTATTGAAAGTAACATGTAAATAGTTCTACATACGTTCTTTTGACATCTTGTTCAATCATTGATCGAAGTTCTTTATCTTGGAAGAATTTGTTCCAAAGACTCTGAAATAAGGAAAACAATCTATTATATAGTCTCACACCTTTGTTTTACTTTTAGTGATTTCAATTTAATAATGTAAATGGTTAAAATTTATTCTTCTCTGAGATCATTTCACATTGCAGATAGAAAACCTGAGACTGGGGTAATTTTTATTAAAATCTAATTTAATCTCAGAAACACATCTTTATTCTAACATCAATTTTTCCAGTTTGATATTATCATATAAAGTCAGCCTTCCTCATCTGCAGGTTCCACAACAAAAATCCAACCAACTGTGGATCAAAAATATTGGGAAAAAATTAAAAATAGCAATACAACAATAAAAAAATACAAATCAGAAAAACAGCACAGTATAACAACTTTATTTAGCATTTACAATCTATTAGGTATTATAAGTAATCTAGAATTAATTCCGTGTATTCTATAGTGTCACCTAAATCGTATGTGTATGATACATAAGGTTATGTATTAATTGTAGCCGCGTTCTAACGACAATATGTACAAGCCTAATTGTGTAGCATCTGGCTTACTGAAGCAGACCCTATCATCTCTCTCGTAAACTGCCGTCAGAGTCGGTTTGGTTGGACGAACCTTCTGAGTTTCTGGTAACGCCGTCCCGCACCCGGAAATGGTCAGCGAACCAATCAGCAGGGTCATCGCTAGCCAGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGAATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTGGACACAAGACAGGCTTGCGAGATATGTTTGAGAATACCACTTTATCCCGCGTCAGGGAGAGGCAGTGCGTAAAAAGACGCGGACTCATGTGAAATACTGGTTTTTAGTGCGCCAGATCTCTATAATCTCGCGCAACCTATTTTCCCCTCGAACACTTTTTAAGCCGTAGATAAACAGGCTGGGACACTTCACATGAGCGAAAAATACATCGTCACCTGGGACATGTTGCAGATCCATGCACGTAAACTCGCAAGCCGACTGATGCCTTCTGAACAATGGAAAGGCATTATTGCCGTAAGCCGTGGCGGTCTGTACCGGGTGCGTTACTGGCGCGTGAACTGGGTATTCGTCATGTCGATACCGTTTGTATTTCCAGCTACGATCACGACAACCAGCGCGAGCTTAAAGTGCTGAAACGCGCAGAAGGCGATGGCGAAGGCTTCATCGTTATTGATGACCTGGTGGATACCGGTGGTACTGCGGTTGCGATTCGTGAAATGTATCCAAAAGCGCACTTTGTCACCATCTTCGCAAAACCGGCTGGTCGTCCGCTGGTTGATGACTATGTTGTTGATATCCCGCAAGATACCTGGATTGAACAGCCGTGGGATATGGGCGTCGTATTCGTCCCGCCAATCTCCGGTCGCTAATCTTTTCAACGCCTGGCACTGCCGGGCGTTGTTCTTTTTAACTTCAGGCGGGTTACAATAGTTTCCAGTAAGTATTCTGGAGGCTGCATCCATGACACAGGCAAACCTGAGCGAAACCCTGTTCAAACCCCGCTTTAAACATCCTGAAACCTCGACGCTAGTCCGCCGCTTTAATCACGGCGCACAACCGCCTGTGCAGTCGGCCCTTGATGGTAAAACCATCCCTCACTGGTATCGCATGATTAACCGTCTGATGTGGATCTGGCGCGGCATTGACCCACGCGAAATCCTCGACGTCCAGGCACGTATTGTGATGAGCGATGCCGAACGTACCGACGATGATTTATACGATACGGTGATTGGCTACCGTGGCGGCAACTGGATTTATGAGTGGGCCCCGGATCTTTGTGAAGGAACCTTACTTCTGTGGTGTGACATAATTGGACAAACTACCTACAGAGATTTAAAGCTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTGTGTATTTTAGATTCCAACCTATGGAACTGATGAATGGGAGCAGTGGTGGAATGCCTTTAATGAGGAAAACCTGTTTTGCTCAGAAGAAATGCCATCTAGTGATGATGAGGCTACTGCTGACTCTCAACATTCTACTCCTCCAAAAAAGAAGAGAAAGGTAGAAGACCCCAAGGACTTTCCTTCAGAATTGCTAAGTTTTTTGAGTCATGCTGTGTTTAGTAATAGAACTCTTGCTTGCTTTGCTATTTACACCACAAAGGAAAAAGCTGCACTGCTATACAAGAAAATTATGGAAAAATATTCTGTAACCTTTATAAGTAGGCATAACAGTTATAATCATAACATACTGTTTTTTCTTACTCCACACAGGCATAGAGTGTCTGCTATTAATAACTATGCTCAAAAATTGTGTACCTTTAGCTTTTTAATTTGTAAAGGGGTTAATAAGGAATATTTGATGTATAGTGCCTTGACTAGAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCAACTGGATAACTCAAGCTAACCAAAATCATCCCAAACTTCCCACCCCATACCCTATTACCACTGCC pHR BackboneAATTACCTGTGGTTTCATTTACTCTAAACCTGTGATTCCTCTGAATTATTTTCATTTTAAAGAAATTGTATTTGTTAAATATGTACTACAAACTTAGTAGTTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAGAACTACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGGTAGAAGAGGCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGAGAGAAGTGTTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTGATATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTATCGATAAGCTTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGAATTACCTGTGGTTTCATTTACTCTAAACCTGTGATTCCTCTGAATTATTTTCATTTTAAAGAAATTGTATTTGTTAAATATGTACTACAAACTTAGTAGTTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAGAACTACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGGTAGAAGAGGCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGAGAGAAGTGTTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTGATATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTATCGATAAGCTTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCGGATCCAAGCTTCAATTGTGGTCACTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGCTCGAGACCTAGAAAAACATGGAGCAATCACAAGTAGCAATACAGCAGCTACCAATGCTGATTGTGCCTGGCTAGAAGCACAAGAGGAGGAGGAGGTGGGTTTTCCAGTCACACCTCAGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGCCTTGACATTATAATAGATTTAGCAGGAATTGAACTAGGAGTGGAGCACACAGGCAAAGCTGCAGAAGTACTTGGAAGAAGCCACCAGAGATACTCACGATTCTGCACATACCTGGCTAATCCCAGATCCTAAGGATTACATTAAGTTTACTAACATTTATATAATGATTTATAGTTTAAAGTATAAACTTATCTAATTTACTATTCTGACAGATATTAATTAATCCTCAAATATCATAAGAGATGATTACTATTATCCCCATTTAACACAAGAGGAAACTGAGAGGGAAAGATGTTGAAGTAATTTTCCCACAATTACAGCATCCGTTAGTTACGACTCTATGATCTTCTGACACAAATTCCATTTACTCCTCACCCTATGACTCAGTCGAATATATCAAAGTTATGGACATTATGCTAAGTAACAAATTACCCTTTTATATAGTAAATACTGAGTAGATTGAGAGAAGAAATTGTTTGCAAACCTGAATAGCTTCAAGAAGAAGAGAAGTGAGGATAAGAATAACAGTTGTCATTTAACAAGTTTTAACAAGTAACTTGGTTAGAAAGGGATTCAAATGCATAAAGCAAGGGATAAATTTTTCTGGCAACAAGACTATACAATATAACCTTAAATATGACTTCAAATAATTGTTGGAACTTGATAAAACTAATTAAATATTATTGAAGATTATCAATATTATAAATGTAATTTACTTTTAAAAAGGGAACATAGAAATGTGTATCATTAGAGTAGAAAACAATCCTTATTATCACAATTTGTCAAAACAAGTTTGTTATTAACACAAGTAGAATACTGCATTCAATTAAGTTGACTGCAGATTTTGTGTTTTGTTAAAATTAGAAAGAGATAACAACAATTTGAATTATTGAAAGTAACATGTAAATAGTTCTACATACGTTCTTTTGACATCTTGTTCAATCATTGATCGAAGTTCTTTATCTTGGAAGAATTTGTTCCAAAGACTCTGAAATAAGGAAAACAATCTATTATATAGTCTCACACCTTTGTTTTACTTTTAGTGATTTCAATTTAATAATGTAAATGGTTAAAATTTATTCTTCTCTGAGATCATTTCACATTGCAGATAGAAAACCTGAGACTGGGGTAATTTTTATTAAAATCTAATTTAATCTCAGAAACACATCTTTATTCTAACATCAATTTTTCCAGTTTGATATTATCATATAAAGTCAGCCTTCCTCATCTGCAGGTTCCACAACAAAAATCCAACCAACTGTGGATCAAAAATATTGGGAAAAAATTAAAAATAGCAATACAACAATAAAAAAATACAAATCAGAAAAACAGCACAGTATAACAACTTTATTTAGCATTTACAATCTATTAGGTATTATAAGTAATCTAGAATTAATTCCGTGTATTCTATAGTGTCACCTAAATCGTATGTGTATGATACATAAGGTTATGTATTAATTGTAGCCGCGTTCTAACGACAATATGTACAAGCCTAATTGTGTAGCATCTGGCTTACTGAAGCAGACCCTATCATCTCTCTCGTAAACTGCCGTCAGAGTCGGTTTGGTTGGACGAACCTTCTGAGTTTCTGGTAACGCCGTCCCGCACCCGGAAATGGTCAGCGAACCAATCAGCAGGGTCATCGCTAGCCAGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGAATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTGGACACAAGACAGGCTTGCGAGATATGTTTGAGAATACCACTTTATCCCGCGTCAGGGAGAGGCAGTGCGTAAAAAGACGCGGACTCATGTGAAATACTGGTTTTTAGTGCGCCAGATCTCTATAATCTCGCGCAACCTATTTTCCCCTCGAACACTTTTTAAGCCGTAGATAAACAGGCTGGGACACTTCACATGAGCGAAAAATACATCGTCACCTGGGACATGTTGCAGATCCATGCACGTAAACTCGCAAGCCGACTGATGCCTTCTGAACAATGGAAAGGCATTATTGCCGTAAGCCGTGGCGGTCTGTACCGGGTGCGTTACTGGCGCGTGAACTGGGTATTCGTCATGTCGATACCGTTTGTATTTCCAGCTACGATCACGACAACCAGCGCGAGCTTAAAGTGCTGAAACGCGCAGAAGGCGATGGCGAAGGCTTCATCGTTATTGATGACCTGGTGGATACCGGTGGTACTGCGGTTGCGATTCGTGAAATGTATCCAAAAGCGCACTTTGTCACCATCTTCGCAAAACCGGCTGGTCGTCCGCTGGTTGATGACTATGTTGTTGATATCCCGCAAGATACCTGGATTGAACAGCCGTGGGATATGGGCGTCGTATTCGTCCCGCCAATCTCCGGTCGCTAATCTTTTCAACGCCTGGCACTGCCGGGCGTTGTTCTTTTTAACTTCAGGCGGGTTACAATAGTTTCCAGTAAGTATTCTGGAGGCTGCATCCATGACACAGGCAAACCTGAGCGAAACCCTGTTCAAACCCCGCTTTAAACATCCTGAAACCTCGACGCTAGTCCGCCGCTTTAATCACGGCGCACAACCGCCTGTGCAGTCGGCCCTTGATGGTAAAACCATCCCTCACTGGTATCGCATGATTAACCGTCTGATGTGGATCTGGCGCGGCATTGACCCACGCGAAATCCTCGACGTCCAGGCACGTATTGTGATGAGCGATGCCGAACGTACCGACGATGATTTATACGATACGGTGATTGGCTACCGTGGCGGCAACTGGATTTATGAGTGGGCCCCGGATCTTTGTGAAGGAACCTTACTTCTGTGGTGTGACATAATTGGACAAACTACCTACAGAGATTTAAAGCTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTGTGTATTTTAGATTCCAACCTATGGAACTGATGAATGGGAGCAGTGGTGGAATGCCTTTAATGAGGAAAACCTGTTTTGCTCAGAAGAAATGCCATCTAGTGATGATGAGGCTACTGCTGACTCTCAACATTCTACTCCTCCAAAAAAGAAGAGAAAGGTAGAAGACCCCAAGGACTTTCCTTCAGAATTGCTAAGTTTTTTGAGTCATGCTGTGTTTAGTAATAGAACTCTTGCTTGCTTTGCTATTTACACCACAAAGGAAAAAGCTGCACTGCTATACAAGAAAATTATGGAAAAATATTCTGTAACCTTTATAAGTAGGCATAACAGTTATAATCATAACATACTGTTTTTTCTTACTCCACACAGGCATAGAGTGTCTGCTATTAATAACTATGCTCAAAAATTGTGTACCTTTAGCTTTTTAATTTGTAAAGGGGTTAATAAGGAATATTTGATGTATAGTGCCTTGACTAGAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCAACTGGATAACTCAAGCTAACCAAAATCATCCCAAACTTCCCACCCCATACCCTATTACCACTGCC cPPT seq SEQ ID NO: 2 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat    60agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc  aaaatttt     118Woodchuck Hepatitus Virus wpre SEQ ID NO: 3aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct    60ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt   120atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg   180tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact   240ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct   300attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg   360ttgggcactg acaattccgt ggtgttgtcg gggaagctga cgtcctttcc atggctgctc   420gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc   480aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt   540cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc tg 592pORF-hIL-12 sequence (5048 bp). hIL-12 open reading frame in bold.Elastin linker is underlined. SEQ ID NO: 4GGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACgtaagtgatatctactagatttatcaaaaagagtgttgacttgtgagcgctcacaattgatacttagattcatcgagagggacacgtcgactactaaccttcttactttcctacagCTGAGATCACCGGCGAAGGAGGGCCACCATGGGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGGTTTTTCTGGCATCTCCCCTCGTGGCCATATGGGAACTGAAGAAAGATGTTTATGTCGTAGAATTGGATTGGTATCCGGATGCCCCTGGAGAAATGGTGGTCCTCACCTGTGACACCCCTGAAGAAGATGGTATCACCTGGACCTTGGACCAGAGCAGTGAGGTCTTAGGCTCTGGCAAAACCCTGACCATCCAAGTCAAAGAGTTTGGAGATGCTGGCCAGTACACCTGTCACAAAGGAGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAAAAAGGAAGATGGAATTTGGTCCACTGATATTTTAAAGGACCAGAAAGAACCCAAAAATAAGACCTTTCTAAGATGCGAGGCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTGACGACAATCAGTACTGATTTGACATTCAGTGTCAAAAGCAGCAGAGGCTCTTCTGACCCCCAAGGGGTGACGTGCGGAGCTGCTACACTCTCTGCAGAGAGAGTCAGAGGGGACAACAAGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGACAGTGCCTGCCCAGCTGCTGAGGAGAGTCTGCCCATTGAGGTCATGGTGGATGCCGTTCACAAGCTCAAGTATGAAAACTACACCAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACCCACCCAAGAACTTGCAGCTGAAGCCATTAAAGAATTCTCGGCAGGTGGAGGTCAGCTGGGAGTACCCTGACACCTGGAGTACTCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAGGTCCAGGGCAAGAGCAAGAGAGAAAAGAAAGATAGAGTCTTCACGGACAAGACCTCAGCCACGGTCATCTGCCGCAAAAATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTATAGCTCATCTTGGAGCGAATGGGCATCTGTGCCCTGCAGT GTTCCTGGAGTAGGGGTACCTGGGGTGGGC GCCAGAAACCTCCCCGTGGCCACTCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCCAAAACCTGCTGAGGGCCGTCAGCAACATGCTCCAGAAGGCCAGACAAACTCTAGAATTTTACCCTTGCACTTCTGAAGAGATTGATCATGAAGATATCACAAAAGATAAAACCAGCACAGTGGAGGCCTGTTTACCATTGGAATTAACCAAGAATGAGAGTTGCCTAAATTCCAGAGAGACCTCTTTCATAACTAATGGGAGTTGCCTGGCCTCCAGAAAGACCTCTTTTATGATGGCCCTGTGCCTTAGTAGTATTTATGAAGACTCGAAGATGTACCAGGTGGAGTTCAAGACCATGAATGCAAAGCTTCTGATGGATCCTAAGAGGCAGATCTTTCTAGATCAAAACATGCTGGCAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAACAGTGAGACTGTGCCACAAAAATCCTCCCTTGAAGAACCGGATTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTCATGCTTTCAGAATTCGGGCAGTGACTATTGATAGAGTGATGAGCTATCTGAATGCTTCCTAAAAAGCGAGGTCCCTCCAAACCGTTGTCATTTTTATAAAACTTTGAAATGAGGAAACTTTGATAGGATGTGGATTAAGAACTAGGGAGGGGGAAAGAAGGATGGGACTATTACATCCACATGATACCTCTGATCAAGTATTTTTGACATTTACTGTGGATAAATTGTTTTTAAGTTTTCATGAATGAATTGCTAAGAAGGGGGGAATTCTTTTGCTTTTTACCCTCGACTAGCTCGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAGATCATTTAAATGTTAATTAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGGATCTCGAGCGGCCGCAATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGAATCGTAACTAACATACGCTCTCCATCAAAACAAAACGAAACAAAACAAACTAGCAAAATAGGCTGTCCCCAGTGCAAGTGCAGGTGCCAGAACATTTCTCTATCGAA pORF-mIL-12 (p35p40) sequence (4846 bp).mIL-12 open reading frame in bold. Elastin linker sequence is undelined.SEQ ID NO: 5GGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACgtaagtgatatctactagatttatcaaaaagagtgttgacttgtgagcgctcacaattgatacttagattcatcgagagggacacgtcgactactaaccttcttactttcctacagCTGAGATCACCGGCGAAGGAGGGCCACCATGGGTCAATCACGCTACCTCCTCTTTTTGGCCACCCTTGCCCTCCTAAACCACCTCAGTTTGGCCAGGGTCATTCCAGTCTCTGGACCTGCCAGGTGTCTTAGCCAGTCCCGAAACCTGCTGAAGACCACAGATGACATGGTGAAGACGGCCAGAGAAAAGCTGAAACATTATTCCTGCACTGCTGAAGACATCGATCATGAAGACATCACACGGGACCAAACCAGCACATTGAAGACCTGTTTACCACTGGAACTACACAAGAACGAGAGTTGCCTGGCTACTAGAGAGACTTCTTCCACAACAAGAGGGAGCTGCCTGCCCCCACAGAAGACGTCTTTGATGATGACCCTGTGCCTTGGTAGCATCTATGAGGACTTGAAGATGTACCAGACAGAGTTCCAGGCCATCAACGCAGCACTTCAGAATCACAACCATCAGCAGATCATTCTAGACAAGGGCATGCTGGTGGCCATCGATGAGCTGATGCAGTCTCTGAATCATAATGGCGAGACTCTGCGCCAGAAACCTCCTGTGGGAGAAGCAGACCCTTACAGAGTGAAAATGAAGCTCTGCATCCTGCTTCACGCCTTCAGCACCCGCGTCGTGACCATCAACAGGGTGATGGGCTATCTGAGCTCCGCC GTTCCTGGAGTAGGGGTACCTGGAGTGGGC GGATCTATGTGGGAGCTGGAGAAAGACGTTTATGTTGTAGAGGTGGACTGGACTCCCGATGCCCCTGGAGAAACAGTGAACCTCACCTGTGACACGCCTGAAGAAGATGACATCACCTGGACCTCAGACCAGAGACATGGAGTCATAGGCTCTGGAAAGACCCTGACCATCACTGTCAAAGAGTTTCTAGATGCTGGCCAGTACACCTGCCACAAAGGAGGCGAGACTCTGAGCCACTCACATCTGCTGCTCCACAAGAAGGAAAATGGAATTTGGTCCACTGAAATTTTAAAAAATTTCAAAAACAAGACTTTCCTGAAGTGTGAAGCACCAAATTACTCCGGACGGTTCACGTGCTCATGGCTGGTGCAAAGAAACATGGACTTGAAGTTCAACATCAAGAGCAGTAGCAGTCCCCCCGACTCTCGGGCAGTGACATGTGGAATGGCGTCTCTGTCTGCAGAGAAGGTCACACTGGACCAAAGGGACTATGAGAAGTATTCAGTGTCCTGCCAGGAGGATGTCACCTGCCCAACTGCCGAGGAGACCCTGCCCATTGAACTGGCGTTGGAAGCACGGCAGCAGAATAAATATGAGAACTACAGCACCAGCTTCTTCATCAGGGACATCATCAAACCAGACCCGCCCAAGAACTTGCAGATGAAGCCTTTGAAGAACTCACAGGTGGAGGTCAGCTGGGAGTACCCTGACTCCTGGAGCACTCCCCATTCCTACTTCTCCCTCAAGTTCTTTGTTCGAATCCAGCGCAAGAAAGAAAAGATGAAGGAGACAGAGGAGGGGTGTAACCAGAAAGGTGCGTTCCTCGTAGAGAAGACATCTACCGAAGTCCAATGCAAAGGCGGGAATGTCTGCGTGCAAGCTCAGGATCGCTATTACAATTCCTCATGCAGCAAGTGGGCATGTGTTCCCTGCAGGGTCCGATCCTAGGATGCAACGGATGCTAGCTCGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAGATCATTTAAATGTTAATTAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGGATCTCGAGCGGCCGCAATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGAATCGTAACTAACATACGCTCTCCATCAAAACAAAACGAAACAAAACAAACTAGCAAAATAGGCTGTCCCCAGTGCAAGTGCAGGTGCCAGAACATTTCTCTATCGAA tmpk sequences <211> 639 <212> DNA <213> Homo sapiensatggcggccc ggcgcggggc tctcatagtg ctggagggcg tggaccgcgc cgggaagagc 60acgcagagcc gcaagctggt ggaagcgctg tgcgccgcgg gccaccgcgc cgaactgctc 120cggttcccgg aaagatcaac tgaaatcggc aaacttctga gttcctactt gcaaaagaaa 180agtgacgtgg aggatcactc ggtgcacctg cttttttctg caaatcgctg ggaacaagtg 240ccgttaatta aggaaaagtt gagccagggc gtgaccctcg tcgtggacag atacgcattt 300tctggtgtgg ccttcaccgg tgccaaggag aatttttccc tagattggtg taaacagcca 360gacgtgggcc ttcccaaacc cgacctggtc ctgttcctcc agttacagct ggcggatgct 420gccaagcggg gagcgtttgg ccatgagcgc tatgagaacg gggctttcca ggagcgggcg 480ctccggtgtt tccaccagct catgaaagac acgactttga actggaagat ggtggatgct 540tccaaaagca tcgaagctgt ccatgaggac atccgcgtgc tctctgagga cgccatccgc 600actgccacag agaagccgct gggggagcta tggaagtga 639 <212> PRT <213>Homo sapiensMet Ala Ala Arg Arg Gly Ala Leu Ile Val Leu Glu Gly Val Asp Arg1               5                   10                  15Ala Gly Lys Ser Thr Gln Ser Arg Lys Leu Val Glu Ala Leu Cys Ala            20                  25                  30Ala Gly His Arg Ala Glu Leu Leu Arg Phe Pro Glu Arg Ser Thr Glu        35                  40                  45Ile Gly Lys Leu Leu Ser Ser Tyr Leu Gln Lys Lys Ser Asp Val Glu    50                  55                  60Asp His Ser Val His Leu Leu Phe Ser Ala Asn Arg Trp Glu Gln Val65                  70                  75                  80Pro Leu Ile Lys Glu Lys Leu Ser Gln Gly Val Thr Leu Val Val Asp                85                  90                  95Arg Tyr Ala Phe Ser Gly Val Ala Phe Thr Gly Ala Lys Glu Asn Phe            100                 105                 110Ser Leu Asp Trp Cys Lys Gln Pro Asp Val Gly Leu Pro Lys Pro Asp        115                 120                 125Leu Val Leu Phe Leu Gln Leu Gln Leu Ala Asp Ala Ala Lys Arg Gly    130                 135                 140Ala Phe Gly His Glu Arg Tyr Glu Asn Gly Ala Phe Gln Glu Arg Ala145                 150                 155                 160Leu Arg Cys Phe His Gln Leu Met Lys Asp Thr Thr Leu Asn Trp Lys                165                 170                 175Met Val Asp Ala Ser Lys Ser Ile Glu Ala Val His Glu Asp Ile Arg            180                 185                 190Val Leu Ser Glu Asp Ala Ile Arg Thr Ala Thr Glu Lys Pro Leu Gly        195                 200                 205 Glu Leu Trp Lys    210 <211> 639 <212> DNA <213> Homo sapiensatggcggccc ggcgcggggc tctcatagtg ctggagggcg tggaccgcgc cgggaagagc 60acgcagagcc gcaagctggt ggaagcgctg tgcgccgcgg gccaccgcgc cgaactgctc 120cggttcccgg aaagatcaac tgaaatcggc aaacttctga gttcctactt gcaaaagaaa 180agtgacgtgg aggatcactc ggtgcacctg cttttttctg caaatcgctg ggaacaagtg 240ccgttaatta aggaaaagtt gagccagggc gtgaccctcg tcgtggacag atacgcattt 300tctggtgtgg ccttcaccgg tgccaaggag aatttttccc tagattggtg taaacagcca 360gacgtgggcc ttcccaaacc cgacctggtc ctgttcctcc agttacagct ggcggatgct 420gccaagcggg gagcgtttgg ccatgagcgc tatgagaacg gggctttcca ggagcgggcg 480ctccggtgtt tccaccagct catgaaagac acgactttga actggaagat ggtggatgct 540tccaaaagca tcgaagctgt ccatgaggac atccgcgtgc tctctgagga cgccatccgc 600actgccacag agaagccgct gggggagcta tggaagtga 639 <211> 212 <212> PRT <213>Homo sapiensMet Ala Ala Arg Arg Gly Ala Leu Ile Val Leu Glu Gly Val Asp Arg1               5                   10                  15Ala Gly Lys Ser Thr Gln Ser Arg Lys Leu Val Glu Ala Leu Cys Ala            20                  25                  30Ala Gly His Arg Ala Glu Leu Leu Arg Phe Pro Glu Arg Ser Thr Glu        35                  40                  45Ile Gly Lys Leu Leu Ser Ser Tyr Leu Gln Lys Lys Ser Asp Val Glu    50                  55                  60Asp His Ser Val His Leu Leu Phe Ser Ala Asn Arg Trp Glu Gln Val65                  70                  75                  80Pro Leu Ile Lys Glu Lys Leu Ser Gln Gly Val Thr Leu Val Val Asp                85                  90                  95Arg Tyr Ala Phe Ser Gly Val Ala Phe Thr Gly Ala Lys Glu Asn Phe            100                 105                 110Ser Leu Asp Trp Cys Lys Gln Pro Asp Val Gly Leu Pro Lys Pro Asp        115                 120                 125Leu Val Leu Phe Leu Gln Leu Gln Leu Ala Asp Ala Ala Lys Arg Gly    130                 135                 140Ala Phe Gly His Glu Arg Tyr Glu Asn Gly Ala Phe Gln Glu Arg Ala145                 150                 155                 160Leu Arg Cys Phe His Gln Leu Met Lys Asp Thr Thr Leu Asn Trp Lys                165                 170                 175Met Val Asp Ala Ser Lys Ser Ile Glu Ala Val His Glu Asp Ile Arg            180                 185                 190Val Leu Ser Glu Asp Ala Ile Arg Thr Ala Thr Glu Lys Pro Leu Gly        195                 200                 205 Glu Leu Trp Lys    210 <211> 636 <212> DNA <213> Homo sapiensatggcggccc ggcgcggggc tctcatagtg ctggagggcg tggaccgcgc cgggaagagc 60acgcagagcc gcaagctggt ggaagcgctg tcgcgcgggc caccgcccga actgctccgg 120ttcccggaaa gatcaactga aatcggcaaa cttctgagtt cctacttgca aaagaaaagt 180gacgtggagg atcactcggt gcacctgctt ttttctgcaa atcgctggga acaagtgccg 240ttaattaagg aaaagttgag ccagggcgtg accctcgtcg tggacagata cgcattttct 300ggtgtggcct tcaccggtgc caaggagaat ttttccctag actggtgtaa acagccagac 360gtgggccttc ccaaacccga cctggtcctg ttcctccagt tacagctggc ggatgctgcc 420aagcggggag cgtttggcca tgagcgctat gagaacgggg ctttccagga gcgggcgctc 480cggtgtttcc accagctcat gaaagacacg actttgaact ggaagatggt ggatgcttcc 540aaaagactcg aagctgtcca tgaggaactc cgcgtgctct ctgaggacgc catccgcact 600gccacagaga agccgctggg ggagctatgg aagtga 636 <211> 211 <212> PRT <213>Homo sapiensMet Ala Ala Arg Arg Gly Ala Leu Ile Val Leu Glu Gly Val Asp Arg1               5                   10                  15Ala Gly Lys Ser Thr Gln Ser Arg Lys Leu Val Glu Ala Leu Ser Arg            20                  25                  30Gly Pro Pro Pro Glu Leu Leu Arg Phe Pro Glu Arg Ser Thr Glu Ile        35                  40                  45Gly Lys Leu Leu Ser Ser Tyr Leu Gln Lys Lys Ser Asp Val Glu Asp    50                  55                  60His Ser Val His Leu Leu Phe Ser Ala Asn Arg Trp Glu Gln Val Pro65                  70                  75                  80Leu Ile Lys Glu Lys Leu Ser Gln Gly Val Thr Leu Val Val Asp Arg                85                  90                  95Tyr Ala Phe Ser Gly Val Ala Phe Thr Gly Ala Lys Glu Asn Phe Ser            100                 105                 110Leu Asp Trp Cys Lys Gln Pro Asp Val Gly Leu Pro Lys Pro Asp Leu        115                 120                 125Val Leu Phe Leu Gln Leu Gln Leu Ala Asp Ala Ala Lys Arg Gly Ala    130                 135                 140Phe Gly His Glu Arg Tyr Glu Asn Gly Ala Phe Gln Glu Arg Ala Leu145                 150                 155                 160Arg Cys Phe His Gln Leu Met Lys Asp Thr Thr Leu Asn Trp Lys Met                165                 170                 175Val Asp Ala Ser Lys Arg Leu Glu Ala Val His Glu Glu Leu Arg Val            180                 185                 190Leu Ser Glu Asp Ala Ile Arg Thr Ala Thr Glu Lys Pro Leu Gly Glu        195                 200                 205 Leu Trp Lys     210<211> 639 <212> DNA <213> Homo sapiensatggcggccc ggcgcggggc tctcatagtg ctggagggcg tggaccgcgc cgggaagagc 60acgcagagcc gcaagctggt ggaagcgctg tgcgccgcgg gccaccgcgc cgaactgctc 120cggttcccgg aaagatcaac tgaaatcggc aaacttctga gttcctactt gcaaaagaaa 180agtgacgtgg aggatcactc ggtgcacctg cttttttctg caaatcgctg ggaacaagtg 240ccgttaatta aggaaaagtt gagccagggc gtgaccctcg tcgtggacag atacgcattt 300tctggtgtgg ccttcaccgg tgccaaggag aatttttccc tagattggtg taaacagcca 360gacgtgggcc ttcccaaacc cgacctggtc ctgttcctcc agttacagct ggcggatgct 420gccaagcggg gagcgtttgg ccatgagcgc tatgagaacg gggctttcca ggagcgggcg 480ctccggtgtt tccaccagct catgaaagac acgactttga actggaagat ggtggatgct 540tccaaaagca tcgaagctgt ccatgaggac atccgcgtgc tctctgagga cgccatccgc 600actgccacag agaagccgct gggggagcta tggaaggac 639 <211> 213 <212> PRT <213>Homo sapiensMet Ala Ala Arg Arg Gly Ala Leu Ile Val Leu Glu Gly Val Asp Arg1               5                   10                  15Ala Gly Lys Ser Thr Gln Ser Arg Lys Leu Val Glu Ala Leu Cys Ala            20                  25                  30Ala Gly His Arg Ala Glu Leu Leu Arg Phe Pro Glu Arg Ser Thr Glu        35                  40                  45Ile Gly Lys Leu Leu Ser Ser Tyr Leu Gln Lys Lys Ser Asp Val Glu    50                  55                  60Asp His Ser Val His Leu Leu Phe Ser Ala Asn Arg Trp Glu Gln Val65                  70                  75                  80Pro Leu Ile Lys Glu Lys Leu Ser Gln Gly Val Thr Leu Val Val Asp                85                  90                  95Arg Tyr Ala Phe Ser Gly Val Ala Phe Thr Gly Ala Lys Glu Asn Phe            100                 105                 110Ser Leu Asp Trp Cys Lys Gln Pro Asp Val Gly Leu Pro Lys Pro Asp        115                 120                 125Leu Val Leu Phe Leu Gln Leu Gln Leu Ala Asp Ala Ala Lys Arg Gly    130                 135                 140Ala Phe Gly His Glu Arg Tyr Glu Asn Gly Ala Phe Gln Glu Arg Ala145                 150                 155                 160Leu Arg Cys Phe His Gln Leu Met Lys Asp Thr Thr Leu Asn Trp Lys                165                 170                 175Met Val Asp Ala Ser Lys Ser Ile Glu Ala Val His Glu Asp Ile Arg            180                 185                 190Val Leu Ser Glu Asp Ala Ile Arg Thr Ala Thr Glu Lys Pro Leu Gly        195                 200                 205 Glu Leu Trp Lys Asp    210 <211> 639 <212> DNA <213> Mus musculusatggcgtcgc gtcggggagc gctcatcgtg ctggagggtg tggaccgtgc tggcaagacc 60acgcagggcc tcaagctggt gaccgcgctg tgcgcctcgg gccacagagc ggagctgctg 120cgtttccccg aaagatcaac ggaaatcggc aagcttctga attcctactt ggaaaagaaa 180acggaactag aggatcactc cgtgcacctg ctcttctctg caaaccgctg ggaacaagta 240ccattaatta aggcgaagtt gaaccagggt gtgacccttg ttttggacag atacgccttt 300tctggggttg ccttcactgg tgccaaagag aatttttccc tggattggtg taaacaaccg 360gacgtgggcc ttcccaaacc tgacctgatc ctgttccttc agttacaatt gctggacgct 420gctgcacggg gagagtttgg ccttgagcga tatgagaccg ggactttcca aaagcaggtt 480ctgttgtgtt tccagcagct catggaagag aaaaacctca actggaaggt ggttgatgct 540tccaaaagca ttgaggaagt ccataaagaa atccgtgcac actctgagga cgccatccga 600aacgctgcac agaggccact gggggagcta tggaaataa 639 <211> 212 <212> PRT <213>Mus musculusMet Ala Ser Arg Arg Gly Ala Leu Ile Val Leu Glu Gly Val Asp Arg1               5                   10                  15Ala Gly Lys Thr Thr Gln Gly Leu Lys Leu Val Thr Ala Leu Cys Ala            20                  25                  30Ser Gly His Arg Ala Glu Leu Leu Arg Phe Pro Glu Arg Ser Thr Glu        35                  40                  45Ile Gly Lys Leu Leu Asn Ser Tyr Leu Glu Lys Lys Thr Glu Leu Glu    50                  55                  60Asp His Ser Val His Leu Leu Phe Ser Ala Asn Arg Trp Glu Gln Val65                  70                  75                  80Pro Leu Ile Lys Ala Lys Leu Asn Gln Gly Val Thr Leu Val Leu Asp                85                  90                  95Arg Tyr Ala Phe Ser Gly Val Ala Phe Thr Gly Ala Lys Glu Asn Phe            100                 105                 110Ser Leu Asp Trp Cys Lys Gln Pro Asp Val Gly Leu Pro Lys Pro Asp        115                 120                 125Leu Ile Leu Phe Leu Gln Leu Gln Leu Leu Asp Ala Ala Ala Arg Gly    130                 135                 140Glu Phe Gly Leu Glu Arg Tyr Glu Thr Gly Thr Phe Gln Lys Gln Val145                 150                 155                 160Leu Leu Cys Phe Gln Gln Leu Met Glu Glu Lys Asn Leu Asn Trp Lys                165                 170                 175Val Val Asp Ala Ser Lys Ser Ile Glu Glu Val His Lys Glu Ile Arg            180                 185                 190Ala His Ser Glu Asp Ala Ile Arg Asn Ala Ala Gln Arg Pro Leu Gly        195                 200                 205 Glu Leu Trp Lys    210 <211> 212 <212> PRT <213> Homo sapiensMet Ala Ala Arg Arg Gly Ala Leu Ile Val Leu Glu Gly Val Asp Arg1               5                   10                  15Ala Gly Lys Ser Thr Gln Ser Arg Lys Leu Val Glu Ala Leu Cys Ala            20                  25                  30Ala Gly His Arg Ala Glu Leu Leu Arg Phe Pro Glu Arg Ser Thr Glu        35                  40                  45Ile Gly Lys Leu Leu Ser Ser Tyr Leu Gln Lys Lys Ser Asp Val Glu    50                  55                  60Asp His Ser Val His Leu Leu Phe Ser Ala Asn Arg Trp Glu Gln Val65                  70                  75                  80Pro Leu Ile Lys Glu Lys Leu Ser Gln Gly Val Thr Leu Val Val Asp                85                  90                  95Arg Tyr Ala Phe Ser Gly Val Ala Tyr Thr Gly Ala Lys Glu Asn Phe            100                 105                 110Ser Leu Asp Trp Cys Lys Gln Pro Asp Val Gly Leu Pro Lys Pro Asp        115                 120                 125Leu Val Leu Phe Leu Gln Leu Gln Leu Ala Asp Ala Ala Lys Arg Gly    130                 135                 140Ala Phe Gly His Glu Arg Tyr Glu Asn Gly Ala Phe Gln Glu Arg Ala145                 150                 155                 160Leu Arg Cys Phe His Gln Leu Met Lys Asp Thr Thr Leu Asn Trp Lys                165                 170                 175Met Val Asp Ala Ser Lys Ser Ile Glu Ala Val His Glu Asp Ile Arg            180                 185                 190Val Leu Ser Glu Asp Ala Ile Arg Thr Ala Thr Glu Lys Pro Leu Gly        195                 200                 205 Glu Leu Trp Lys    210 <211> 214 <212> PRT <213> Homo sapiensMet Ala Ala Arg Arg Gly Ala Leu Ile Val Leu Glu Gly Val Asp Gly1               5                   10                  15Ala Gly Lys Ser Thr Gln Ser Arg Lys Leu Val Glu Ala Leu Cys Ala            20                  25                  30Ala Gly His Arg Ala Glu Leu Leu Arg Phe Pro Glu Arg Ser Thr Glu        35                  40                  45Ile Gly Lys Leu Leu Ser Ser Tyr Leu Gln Lys Lys Ser Asp Val Glu    50                  55                  60Asp His Ser Val His Leu Leu Phe Ser Ala Asn Arg Trp Glu Gln Val65                  70                  75                  80Pro Leu Ile Lys Glu Lys Leu Ser Gln Gly Val Thr Leu Val Val Asp                85                  90                  95Arg Tyr Ala Phe Ser Gly Val Ala Phe Thr Gly Ala Lys Glu Asn Phe            100                 105                 110Ser Leu Asp Trp Cys Lys Gln Pro Asp Val Gly Leu Pro Lys Pro Asp        115                 120                 125Leu Val Leu Phe Leu Gln Leu Thr Pro Glu Val Gly Leu Lys Arg Ala    130                 135                 140Arg Ala Arg Gly Gln Leu Asp Arg Tyr Glu Asn Gly Ala Phe Gln Glu145                 150                 155                 160Arg Ala Leu Arg Cys Phe His Gln Leu Met Lys Asp Thr Thr Leu Asn                165                 170                 175Trp Lys Met Val Asp Ala Ser Lys Ser Ile Glu Ala Val His Glu Asp            180                 185                 190Ile Arg Val Leu Ser Glu Asp Ala Ile Ala Thr Ala Thr Glu Lys Pro        195                 200                 205Leu Gly Glu Leu Trp Lys     210 

1.-23. (canceled)
 24. A method of expressing IL-12 in a cell that issecreted above a threshold level, optionally a cancer cell, comprising:contacting the cell with a composition comprising a vector construct oran isolated virus comprising a lentiviral delivery vector and an IL-12expression cassette, wherein the lentiviral delivery vector comprises acentral polypurine tract (cPPT) sequence, optionally SEQ ID NO: 2 and/ora woodchuck hepatitis virus post-transcriptional regulatory element(WPRE), optionally SEQ ID NO: 3, under conditions that permittransduction of the cell, thereby providing a transduced cell, whereinthe IL-12 is secreted and the IL-12 secreted per cell is above athreshold level.
 25. The method of claim 24, further comprising a stepof isolating the transduced cell or isolating a population of cellscomprising the transduced cell.
 26. The method of claim 24, furthercomprising: a. growth arresting the transduced cell or the population ofcells; and b. optionally introducing the transduced growth arrestedcell, population of cells and/or a composition comprising saidtransduced growth arrested cell or population of cells in a subject.27.-29. (canceled)
 30. The method of claim 31, wherein the cancer isleukemia, optionally ALL, AML, CML or CLL.
 31. A method of inducing orenhancing an immune response, optionally a memory immune response in asubject, optionally with cancer or an increased risk of cancer and/ortreating a subject with cancer or an increased risk of cancer comprisingadministering: i) an isolated virus-comprising a lentiviral deliveryvector and an IL-12 expression cassette wherein the lentiviral vectorcomprises a central polypurine tract (cPPT) sequence, optionally SEQ IDNO: 2 and/or a woodchuck hepatitis virus post-transcriptional regulatoryelement (WPRE), optionally SEQ ID NO: 3, ii) a transduced cellcomprising a lentiviral delivery vector and an IL-12 expression cassettewherein the transduced cell expresses IL-12 above a threshold level iii)a population of cells comprising the transduced cell of ii), or iv) acomposition comprising i), ii) and/or iii).
 32. (canceled) 33.(canceled)
 34. The method of claim 31 wherein the transduced cell isgrowth arrested prior to administering to the subject, optionallywherein the transduced cell is irradiated prior to administering to thesubject.
 35. (canceled)
 36. A method of delivering IL-12 to a subject,optionally with cancer or an increased risk or cancer, optionally, forenhancing cancer treatment comprising: a. generating a recombinant IL-12secreting cell wherein the IL-12 secreted per cell is above a thresholdlevel; and b. introducing an effective number of the generatedrecombinant IL-12 secreting cells to the subject, optionally in apopulation of cells comprising the recombinant IL-12 secreting cell. 37.(canceled)
 38. (canceled)
 39. The method of claim 36 wherein thethreshold level of IL-12 secreted is at least 1.5 fg/ml cells/2 hrs ofIL-12, at least 1.5 pg/ml cells/2 hrs of IL-12, or at least 1500pg/mL/10⁶ cells/2 hrs of IL-12, optionally at least 1500 pg/mL/10⁶cells/2 hrs, 1500-2500 pg/mL/10⁶ cells/2 hrs, 2500-5000 pg/mL/10⁶cells/2 hrs, 5000-7500 pg/mL/10⁶ cells/2 hrs, 7500-10000 pg/mL/10⁶cells/2 hrs, 10000-12500 pg/mL/10⁶ cells/2 hrs, 12500-15000 pg/mL/10⁶cells/2 hrs, 15000-17500 pg/mL/10⁶ cells/2 hrs, 17500-20000 pg/mL/10⁶cells/2 hrs or at least 20000 pg/mL/10⁶ cells/2 hrs of IL-12.
 40. Themethod of claim 36, wherein the recombinant IL-12 secreting cell isgenerated by contacting the cell with a composition comprising alentiviral delivery vector and an IL-12 expression cassette, wherein thelentiviral delivery vector comprises a central polypurine tract (cPPT)sequence, optionally SEQ ID NO: 2 and/or a woodchuck hepatitis viruspost-transcriptional regulatory element (WPRE), optionally SEQ ID NO: 3.41. The method of claim 36, wherein the recombinant cell is a cancercell, optionally derived from the subject with cancer.
 42. The method ofclaim 36, wherein the recombinant cell or population of cells areintroduced by IP injection, subcutaneously or intradermally.
 43. Themethod of claim 36, wherein the cancer is a leukemia, optionally ALL,AML, CML or CLL.
 44. The method of claim 36, wherein the IL-12 secretingcell is growth arrested prior to introducing to the subject. 45.(canceled)
 46. (canceled)
 47. The method of claim 31, wherein the immuneresponse leads to long-term immune memory and/or does not induce orenhance antagonistic cytokines, and/or wherein the response is initiatedsubstantially free of inducing or enhancing of a CD8⁺ T cell-dependentimmune response.
 48. (canceled)
 49. The method of claim 31 wherein thelevel of IL-12 secreted is above a threshold level that enhancesdendritic cell maturation and/or antigen presentation. 50.-71.(canceled)
 72. The method of claim 36, wherein the number of cellsintroduced ranges from 10⁵ cells to 10⁹ cells, optionally about 10⁵,about 10⁶ cells, about 10⁷ cells, about 10⁸ cells, or about 10⁹ cells.73. (canceled)
 74. The method of claim 36 wherein the population ofcells optionally comprises at least 0.1 to 1% IL-12 producing leukemiccells, optionally about 0.5%, about 1%, about 1-5%, 5-10%, 10-20% ormore IL-12 producing leukemic cells, and wherein the population of cellssecretes above the threshold level optionally the threshold levelnecessary to induce or enhance a CD4+ T cell dependent immune response,optionally at least 1500 pg/mL/10⁶ cells/2 hrs, 1500-2500 pg/mL/10⁶cells/2 hrs, 2500-5000 pg/mL/10⁶ cells/2 hrs, 5000-7500 pg/mL/10⁶cells/2 hrs, 7500-10000 pg/mL/10⁶ cells/2 hrs, 10000-12500 pg/mL/10⁶cells/2 hrs, 12500-15000 pg/mL/10⁶ cells/2 hrs, 15000-17500 pg/mL/10⁶cells/2 hrs, 17500-20000 pg/mL/10⁶ cells/2 hrs or at least 20000pg/mL/10 cells/2 hrs of IL-12.
 75. The method of claim 24, wherein theIL-12 expression cassette comprises a polynucleotide optionally encodinga p35 polypeptide and a polynucleotide encoding a p40 polypeptide; or apolynucleotide encoding an IL-12 fusion polypeptide.
 76. The method ofclaim 75 wherein the IL-12 fusion polypeptide has at least 70% sequenceidentity to SEQ ID NO: 4 and binds an IL-12 receptor.
 77. The method ofclaim 24 wherein the vector construct further comprises one or more of:an activator polynucleotide encoding a polypeptide that converts aprodrug to a drug, optionally a modified tmpk polynucleotide; adetection cassette, optionally a CD19, truncated CD19, CD20, human CD24,murine HSA, human CD25 (huCD25), a truncated form of low affinity nervegrowth factor receptor (LNGFR), truncated CD34, eGFP, eYFP, or any otherfluorescent protein or erythropoietin receptor (EpoR) polynucleotide; ora polynucleotide with at least 70% sequence identity to saidpolynucleotide; and an immune modulatory cassette, optionally apolynucleotide that encodes a polypeptide that modulates an immune cell,optionally a dendritic cell or a T cell, optionally a CD4+ T cell,optionally CD40L, IL-7, or IL-15.